Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches
Edited by Susan S. Taylor, University of California, San Diego, La Jolla, CA, and approved August 16, 2017 (received for review February 2, 2017)
Significance
Protein phosphorylation by cAMP-dependent protein kinase (PKA) triggers cellular changes, including fight-or-flight responses in heart cells, and synaptic potentiation in neurons. Uncontrolled activity of PKA catalytic subunits is pathological; however, the mechanism for directing PKA in cells is unclear. Using an approach for monitoring cellular PKA subunit interactions, we show that—contrary to recent proposals—catalytic subunits are released from regulatory subunits by cAMP. Instead, we identify mechanisms for rapid recapture of liberated catalytic subunits. Regulatory subunits are expressed much more highly than catalytic subunits to support rapid catalytic subunit reassociation. Furthermore, analysis of global PKA architecture reveals that type II regulatory subunit anchoring is compatible with catalytic subunit release and recapture within the cell membrane.
Abstract
Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
Protein kinase A (PKA), also known as cAMP-dependent protein kinase, is the major intracellular receptor for the second messenger cAMP (1). Activation of PKA by cAMP underlies responses throughout the body, including sympathetic regulation of the heart downstream of β-adrenergic receptor (β-AR) activation (2) and changes in the strength of synaptic connections between neurons (3). PKA consists of regulatory (R) subunit constitutive dimers that sequester catalytic (C) subunits before cAMP activation. There are two types of R subunit. Type II (RII) subunits associate with the low-speed particulate fraction after tissue homogenization, whereas RI subunits do not (4). This results from anchoring of RII but generally, not RI at subcellular sites that include the cell membrane by A-kinase anchoring proteins (AKAPs) (2). Imbalances in the expression or activity of R (5) and C (6) subunits or disruptions in PKA anchoring (7) lead to disease. An enduring challenge in cAMP/PKA research is to understand how PKA is directed to its cellular substrates. Experiments using fluorescent reporters have confirmed that elevations in cAMP concentration and PKA activity are localized within the cell (8, 9), with PKA activity clustering close to anchoring sites (9). However, the mechanism for restraining C subunits after their release is still unclear.
After activation of an R–C complex by cAMP, the range over which the C subunit can phosphorylate substrates will depend on its rate of diffusion and the rate of recapture by R subunits. New potential mechanisms for reducing diffusion and increasing C-subunit recapture have been investigated in recent years. N-terminal myristylation is thought to restrict some free C subunits to the intracellular face of the membrane bilayer (10), slowing their diffusion velocity (11) and restricting their activity to the plane of the membrane. Consistent with this model, RII but not RI subunits increase binding of myristylated C subunits to liposomes (12) probably by stabilizing the “myr-out” conformation of the myristylated C-subunit A helix (13, 14). Many AKAPs also localize to the cell and organellar membranes (15). AKAPs present amphipathic helices that bind to the dimerization and docking (D/D) domain formed by the first 45 amino acids of RII subunits (16–18). However, the first ∼100 amino acids of RII are not visible in the electron density of the most complete crystal structures of RII–C (13, 19), and therefore, it is not clear whether association with membrane-tethered AKAPs is compatible with inserting C subunits into the membrane.
An alternative proposed mechanism for limiting C-subunit diffusion is that C subunits are never released from RII subunits but instead, access nearby substrates while tethered to RII on cAMP activation (20, 21). This theory was supported by experiments showing no effect of β-AR stimulation on C-subunit coprecipitation with anchored RII subunits (20, 21). However, this experiment does not exclude the possibility that R and C subunits reassociate during coimmunoprecipitation after cell lysis. This possibility could not be excluded, as before our study, there has been no method to monitor association of endogenous PKA subunits in cells with temporal precision. Final aspects of PKA that could support rapid R- and C-subunit association are the stoichiometry and concentrations of its subunits. PKA is unusual among the protein kinase A, G, C (AGC) group in that its regulatory and catalytic elements are formed by separate polypeptides (22). Binding studies show that increasing the concentration of RI subunits, with fixed C-subunit concentration, increases the fraction of C subunits bound to R subunits, even in high concentrations of cAMP (23). This means that higher R-subunit concentration increases the rate of R–C complex formation irrespective of cAMP concentration. Surprisingly, little information is available regarding the concentrations of endogenous PKA subunits.
We used a three-pronged approach to investigate mechanisms for restraining C subunits. This combined approach has enabled us to more clearly define which mechanisms do—and do not—contribute to restraining C subunits.
Results
Light-Activated Cross-Linking Indicates That C Subunits Dissociate from R Subunits in Cells Following β-AR Stimulation.
To investigate the notion that C subunits remain tethered to R subunits in cells during cAMP elevation (20, 21), we developed an approach that uses the UV light-activated cross-linker succinimidyl 4,4′-azipentanoate (SDA). We focused on human embryonic kidney (HEK) cells, which are a model cell line for studying cAMP/PKA signaling (24). An overview of this process is shown in Fig. 1 A–C. Cells are first incubated with membrane-permeable SDA, leading to covalent attachment at primary amines including lysine side chains on RI (red in Fig. 1A), RII (blue in Fig. 1A), and C (green in Fig. 1A) subunits. During 365-nm illumination, the diazirine moiety of SDA may react with nearby molecules, leading to covalent cross-links (Fig. 1B). Since the SDA spacer arm is 3.9 Å, R–C cross-linking is strongly favored when the two proteins are associated (Fig. 1B). After cell lysis, either RI subunits or RII subunits are precipitated using immobilized GST fusions (Fig. 1C) to the RI-anchoring disruptor (RIAD) sequence (25) or the C-terminal 93 amino acids of RII-selective AKAP79 (AKAP79c93). R-subunit precipitation is performed with cAMP, and therefore, C-subunit coprecipitation only occurs through R–C cross-linking. The efficacy of this approach is shown for HEK293T cells in Fig. 1D. GST alone precipitated neither RI nor RII (Fig. 1D, lanes 2 and 3), whereas GST-RIAD (Fig. 1D, lanes 4 and 5), and GST-AKAP79c93 (Fig. 1D, lanes 6 and 7) pulled down either RI or RII, respectively. Control experiments confirmed that GST-RIAD pulls down RIα and RIβ with similar efficiency (Fig. S1 A and B). UV exposure led to coprecipitation of covalently linked C subunits, visible as a band at 90 kDa [Fig. 1D, immunoblot (IB):C] corresponding to either 1RI-1C (Fig. 1D, lane 5) or 1RII-1C (Fig. 1D, lane 7) cross-linked heterodimers.
Fig. 1.
Fig. S1.
We applied this approach to determine if RII and C subunits remain associated when β-ARs are stimulated with isoproterenol. We incubated SDA-treated HEK293T cells for 5 min with either 1 μM isoproterenol or vehicle. UV light illumination was either performed immediately or delayed until after cell lysis. Anti-C IB signal intensity at ∼90 kDa after RII subunit pull down shows how much C subunit was bound to RII during UV illumination (indicated by the arrow in Fig. 1E). C–RII association was reduced after isoproterenol stimulation (Fig. 1E, lanes 2 and 3) by 78 ± 7% (P = 0.0015, n = 4) according to densitometry (Fig. 1F, prelysis). However, C subunits were found to have returned to RII after lysis: when UV light illumination was delayed until postlysis (Fig. 1E, lanes 4 and 5), isoproterenol led to a small increase in RII–C cross-linking (27 ± 3%, P = 0.0016, n = 4) (Fig. 1F). Reassociation was abolished when we supplemented lysis buffer with 10 μM exogenous cAMP 10 min before UV-induced cross-linking postlysis (Fig. S1 C and D). We also performed experiments in which we coupled SDA cross-linking to GST-RIAD pull down. These experiments showed that C subunits also dissociate from RI on isoproterenol stimulation (Fig. S1 E and F). Together, our experiments using SDA cross-linking show that strong β-AR stimulation triggers C-subunit dissociation from both RI and RII subunits in cells and that coprecipitation of C in anti-RII immune complexes after isoproterenol stimulation (20, 21) likely occurs because of dilution of cAMP after cell lysis.
RI and RII Subunits Are in Large Excess of C Subunits.
The results of our SDA cross-linking experiments with live cells led us to consider alternative mechanisms for restricting C-subunit activity. We next used quantitative immunoblotting to explore the possibility that R subunits outnumber C subunits. We took advantage of highly specific antibodies, which we independently validated (Fig. S2), to detect RI, C, and either the α or β isoforms of RII subunits. Our approach is exemplified for analysis of homogenates extracted from HEK293T cells (Fig. 2). HEK293T cell extract was run alongside reference concentrations of highly purified C (Fig. 2A, row 1), RI (Fig. 2A, row 2), RIIα (Fig. 2A, row 3), or RIIβ (Fig. 2A, row 4). Antibody binding was determined using a chemiluminescent substrate in tandem with a charge-coupled device imager, and intensity at reference subunit concentrations was used to fit Hill function calibration curves for C (Fig. 2B, Upper Left), RI (Fig. 2B, Upper Right), RIIα (Fig. 2B, Lower Left), and RIIβ (Fig. 2B, Lower Right). All protein extracts were quantified using this approach (Fig. 2B and Fig. S3). In the case of HEK293T cells, it was possible to approximate subunit copy numbers per cell (Fig. 2C and Table S1). Numbers from four independent sets of experiments revealed that there are ≈2.42 ± 0.21 × 105 C, 7.34 ± 0.13 × 105 RI, 15.4 ± 0.23 × 105 RIIα, and 3.00 ± 0.69 × 105 RIIβ subunits per cell (Fig. 2C and Table S1). These copy numbers are the same order of magnitude as the GTPase Ras (26), another wide-acting signaling protein. HEK293 cell volume has been determined by different methods (27, 28) to be ∼2 pL per cell. This equates to cellular PKA subunit concentrations of ∼1.5 μM for RII, ∼0.6 μM for RI, and ∼0.2 μM for C subunits.
Fig. 2.
Fig. S2.
Fig. S3.
Table S1.
| PKA subunit | Fraction of HEK293T cell extract (ppm) | Subunit molecular mass (Da) | Copies per 1 g extract (×1018) | Copies per cell (×106) |
|---|---|---|---|---|
| C | 56.6 ± 4 | 40,590* | 0.840 ± 0.06 | 0.242 ± 0.021 |
| RI | 183 ± 34 | 43,028† | 2.56 ± 0.48 | 0.734 ± 0.13 |
| RIIα | 409 ± 72 | 45,518 | 5.41 ± 0.95 | 1.54 ± 0.23 |
| RIIβ | 82.9 ± 21 | 46,302 | 1.08 ± 0.27 | 0.300 ± 0.069 |
Copy numbers were determined according to the formula listed in SI Materials and Methods.
*
The molecular mass of Cα was used, since this is the predominant isoform in HEK293T cells according to the molecular weight detected with Pan C antibody (e.g., Fig. 1D).
†
Average of RIα (42,982 Da) and RIβ (43,073 Da).
To investigate whether this uneven subunit ratio is a general feature of PKA signaling, we analyzed protein extracts collected from Sprague–Dawley rats. Brain tissue was extracted and separated into forebrain and cerebellum, and a fraction enriched in nerve endings was also collected from forebrain. Additional extracts were prepared from heart, liver, lungs, and skeletal muscle. An identical protein extraction method was used in all cases. In sum, subunit concentrations were determined in protein extracts from eight different sample types (Fig. S3). Average concentrations (n = 4) are shown in nanograms per milligram protein extract in Fig. 3A. PKA subunit concentrations are relatively high in forebrain, with RIIβ accounting for 0.29% total forebrain protein, and combined forebrain PKA subunits constitute 0.41% forebrain protein (Fig. 3A). These concentrations are approaching those of calmodulin-dependent protein kinase II, which constitutes 0.86% cerebral cortex (29), and calcineurin, which is ∼1% of cerebral cortical and hippocampal protein (30).
Fig. 3.
We next calculated copy numbers of RI (Fig. 3B, red), RIIα (Fig. 3B, light blue), and RIIβ (Fig. 3B, dark blue) relative to C subunits (Fig. 3B, green) in each extract type. In all cases, R subunits greatly outnumber C subunits, with a combined average of 17.2 ± 1.7-fold more R than C subunits (P = 4.8 × 10−6) (Fig. 3B). When the average copy numbers are compared by Student’s t test in each extract, both RI and RII separately outnumber C subunits, with P < 0.05 in every case except RI in liver, in which the comparison is underpowered (P = 0.051). R subunits are in greater than 20-fold excess of C subunits in forebrain, cerebellum, and lungs. A combined analysis of all ratios reveals that RII subunits typically outnumber RI subunits by ∼2:1. Cerebellum and heart are notable exceptions to this rule. The approximately twofold higher expression of RI than RII in cerebellum may be related to the different (presynaptic) role that PKA plays in synaptic plasticity in cerebellar granule cells (31, 32). The elevated expression of the β isoform of RII in brain extracts is consistent with previous reports (33). Overall, our stoichiometric analysis reveals that C subunits are greatly outnumbered by R subunits across tissue types.
Insights into Anchored Type II PKA Holoenzyme Structure from Cross-Linking Coupled to MS.
We next aimed to resolve uncertainties in the structure of anchored RII–C complexes in an effort to determine whether anchoring of RII subunits—the predominant R-subunit isoform in most organs (Fig. 3B)—is compatible with release and capture of C subunits in cellular membranes. There are two conceivable positions for the D/D domain within the RIIβ–C complex: between the N lobes of the C subunits or between the second cyclic nucleotide binding (CNBB) domains of the RII subunits (13, 19). To resolve this uncertainty, we turned to cross-linking coupled to MS (XL-MS), which is a rapidly developing technique suited to structural investigation of large dynamic multiprotein complexes (34). In XL-MS, protein samples are cross-linked and digested into peptides, and the sequence of cross-linked peptides is determined by MS. Cross-links reveal which regions of the protein complex are close in space. We used the homobifunctional cross-linker disuccinimidyl suberate (DSS), which links amines, including those at the termini of lysine side chains, with a maximum span of ∼30 Å (34). We first cross-linked PKA holoenzymes comprising RIIβ–Cβ either alone or in complex with AKAP18α. AKAP18α possesses typical AKAP properties that make it a suitable prototype for studying PKA structure (18, 35), including lipid modification sites in its first six amino acids that enable membrane insertion (36). Samples were imaged using Coomassie staining, immunoblotting, and RII overlay after electrophoresis either before (odd-numbered lanes in Fig. 4A) or after (even lanes in Fig. 4A) DSS incubation. Cross-linking of RIIβ–Cβ led to prominent bands at ∼90 and 140 kDa, with a weaker band at ∼180 kDa. Anti-C (Fig. 4A, lane 6) and anti-RIIβ (Fig. 4A, lane 10) IBs indicate that these three species represent 2RII, 2RII–1C, and 2RII–2C. Inclusion of the AKAP shifts all three bands higher (lanes 4, 8, and 12 in Fig. 4A) by approximately the mass of AKAP18α. RII overlay confirmed that AKAP18α was effectively cross-linked within the complex (Fig. 4A, lane 16). Sequences of cross-linked peptides identified in the RIIβ–Cβ and AKAP18α–RIIβ–Cβ samples are listed in Tables S2 and S3, respectively. A total of 126 cross-linked peptides were identified after cross-linking of RIIβ–Cβ, including 42 R–R intralinks, 74 C–C intralinks, and 10 R–C interlinks. Fig. 4B shows the overall pattern of intralinks (Fig. 4B, purple) and interlinks (Fig. 4B, black) after cross-linking RIIβ (Fig. 4B, blue) and Cβ (Fig. 4B, green) in the absence of AKAP18α.
Fig. 4.
Table S2.
| Cross-linked peptide sequence | Protein 1 | Protein 2 | Position 1 | Position 2 | deltaS | Linear discriminant score |
|---|---|---|---|---|---|---|
| FPSHFSSDLKDLLR-KGTAR-a10-b1 | Cβ | RIIβ | 266 | 46 | 0.36 | 49.45 |
| RKGKSEVEENGAVEIAR-GQYFGELALVTNKPR-a2-b13 | RIIβ | RIIβ | 326 | 357 | 0 | 46.95 |
| IIHPKTDDQR-LKVVDVIGTK-a5-b2 | RIIβ | RIIβ | 135 | 285 | 0.67 | 46.2 |
| RKGKSEVEENGAVEIAR-GQYFGELALVTNKPR-a4-b13 | RIIβ | RIIβ | 328 | 357 | 0 | 45.47 |
| LKQIEHTLNEK-ILDKQK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.74 | 45.25 |
| KGKSEVEENGAVEIAR-GQYFGELALVTNKPR-a3-b13 | RIIβ | RIIβ | 328 | 357 | 0 | 45.13 |
| GKSEVEENGAVEIAR-GQYFGELALVTNKPR-a2-b13 | RIIβ | RIIβ | 328 | 357 | 0 | 45.12 |
| ATEQYYAMKILDK-VMLVKHK-a9-b5 | Cβ | Cβ | 72 | 61 | 0.57 | 44.43 |
| KMYESFIESLPFLK-IIVKNNAK-a1-b4 | RIIβ | RIIβ | 263 | 256 | 0 | 44 |
| KGKSEVEENGAVEIAR-ITMKR-a1-b4 | RIIβ | RIIβ | 326 | 324 | 0.93 | 43.45 |
| KGKSEVEENGAVEIAR-ITMKR-a3-b4 | RIIβ | RIIβ | 328 | 324 | 0.93 | 43.41 |
| NLLQVDLTKR-IVSGKVR-a9-b5 | Cβ | Cβ | 279 | 254 | 0.47 | 43.36 |
| IIHPKTDDQR-IIHPKTDDQR-a5-b5 | RIIβ | RIIβ | 135 | 135 | 0 | 43.03 |
| NGVSDIKTHK-AKEDFLR-a7-b2 | Cβ | Cβ | 292 | 23 | 0.72 | 42.73 |
| KMYESFIESLPFLK-KGTAR-a1-b1 | RIIβ | RIIβ | 263 | 46 | 0 | 42.2 |
| LKVVDVIGTK-ITMKR-a2-b4 | RIIβ | RIIβ | 285 | 324 | 0 | 41.89 |
| KMYESFIESLPFLK-NNAKKR-a1-b4 | RIIβ | RIIβ | 263 | 260 | 0 | 41.6 |
| ATEQYYAMKILDK-VVKLK-a9-b3 | Cβ | Cβ | 72 | 81 | 0.48 | 40.88 |
| KGKSEVEENGAVEIAR-KGTAR-a3-b1 | RIIβ | RIIβ | 328 | 46 | 0.45 | 40.87 |
| RKMYESFIESLPFLK-IIVKNNAK-a2-b4 | RIIβ | RIIβ | 263 | 256 | 0.79 | 40.73 |
| FPSHFSSDLKDLLR-AKEDFLR-a10-b2 | Cβ | Cβ | 266 | 23 | 0.58 | 40.68 |
| IIHPKTDDQR-KGTAR-a5-b1 | RIIβ | RIIβ | 135 | 46 | 0 | 40.51 |
| LVKDGEHVIDQGDDGDNFYVIDR-KGTAR-a3-b1 | RIIβ | RIIβ | 174 | 46 | 0 | 40.26 |
| LKQIEHTLNEKR-ILDKQK-a11-b4 | Cβ | Cβ | 92 | 76 | 0 | 40.01 |
| KGKSEVEENGAVEIAR-GQYFGELALVTNKPR-a1-b13 | RIIβ | RIIβ | 326 | 357 | 0 | 39.45 |
| GKSEVEENGAVEIAR-ITMKRK-a2-b4 | RIIβ | RIIβ | 328 | 324 | 0.94 | 39.18 |
| RKMYESFIESLPFLK-NNAKK-a2-b4 | RIIβ | RIIβ | 263 | 260 | 0.95 | 38.93 |
| KGKSEVEENGAVEIAR-KGTAR-a1-b1 | RIIβ | RIIβ | 326 | 46 | 0.38 | 38.92 |
| LKQIEHTLNEKR-AKEDFLR-a2-b2 | Cβ | Cβ | 83 | 23 | 0 | 38.7 |
| LKVVDVIGTK-KGTAR-a2-b1 | RIIβ | RIIβ | 285 | 46 | 0 | 38.35 |
| KGSEVESVKEFLAK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 16 | 292 | 0.93 | 38.18 |
| KKTLGTGSFGR-VMLVKHK-a2-b5 | Cβ | Cβ | 47 | 61 | 0 | 38.12 |
| LKVVDVIGTK-IIVKNNAK-a2-b4 | RIIβ | RIIβ | 285 | 256 | 0 | 37.95 |
| IIHPKTDDQR-IIVKNNAK-a5-b4 | RIIβ | RIIβ | 135 | 256 | 0 | 37.55 |
| KKTLGTGSFGR-VEAPFIPKFR-a2-b8 | Cβ | Cβ | 47 | 317 | 0.9 | 37.47 |
| HKATEQYYAMKILDKQK-VVKLK-a11-b3 | Cβ | Cβ | 72 | 81 | 0.74 | 37.44 |
| FGNLKNGVSDIKTHK-AKEDFLRK-a12-b2 | Cβ | Cβ | 292 | 23 | 0 | 37.21 |
| FGNLKNGVSDIKTHK-AKEDFLR-a12-b2 | Cβ | Cβ | 292 | 23 | 0.27 | 37.12 |
| KKTLGTGSFGR-KVEAPFIPKFR-a2-b9 | Cβ | Cβ | 47 | 317 | 0.77 | 36.83 |
| KMYESFIESLPFLK-NNAKK-a1-b4 | RIIβ | RIIβ | 263 | 260 | 0 | 36.51 |
| LKVVDVIGTK-RVKGR-a2-b3 | RIIβ | Cβ | 285 | 192 | 0 | 36.35 |
| NGVSDIKTHK-AKEDFLRK-a7-b2 | Cβ | Cβ | 292 | 23 | 0.76 | 36.21 |
| HKATEQYYAMKILDK-LKQIEHTLNEKR-a11-b2 | Cβ | Cβ | 72 | 83 | 0.26 | 36.19 |
| GKSEVEENGAVEIAR-ITMKR-a2-b4 | RIIβ | RIIβ | 328 | 324 | 0.27 | 36.19 |
| ILDKQK-VVKLK-a4-b3 | Cβ | Cβ | 76 | 81 | 0.89 | 36.05 |
| LKQIEHTLNEKR-QKVVK-a2-b2 | Cβ | Cβ | 83 | 78 | 0 | 36.05 |
| LKQIEHTLNEKR-ILDKQK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.73 | 36.04 |
| LKQIEHTLNEK-ILDKQKVVK-a2-b6 | Cβ | Cβ | 83 | 78 | 0.87 | 35.74 |
| LKVVDVIGTK-NNAKK-a2-b4 | RIIβ | RIIβ | 285 | 260 | 0.79 | 35.65 |
| KKTLGTGSFGR-KVEAPFIPKFR-a1-b9 | Cβ | Cβ | 46 | 317 | 0.71 | 35.1 |
| LVKDGEHVIDQGDDGDNFYVIDR-IIVKNNAK-a3-b4 | RIIβ | RIIβ | 174 | 256 | 0.18 | 35 |
| HKATEQYYAMKILDK-LKQIEHTLNEK-a11-b2 | Cβ | Cβ | 72 | 83 | 0.75 | 34.9 |
| LVKDGEHVIDQGDDGDNFYVIDR-IVSGKVR-a3-b5 | RIIβ | Cβ | 174 | 254 | 0.32 | 34.82 |
| GQYFGELALVTNKPR-ITMKR-a13-b4 | RIIβ | RIIβ | 357 | 324 | 0 | 34.79 |
| NGVSDIKTHKWFATTDWIAIYQR-AKEDFLR-a10-b2 | Cβ | Cβ | 295 | 23 | 0.26 | 34.64 |
| RKMYESFIESLPFLK-ILDKQK-a2-b4 | RIIβ | Cβ | 263 | 76 | 0.69 | 34.62 |
| GQYFGELALVTNKPR-KGTAR-a13-b1 | RIIβ | RIIβ | 357 | 46 | 0 | 34.42 |
| QIEHTLNEKR-ILDKQK-a9-b4 | Cβ | Cβ | 92 | 76 | 0 | 34.25 |
| LKQIEHTLNEKR-ILDKQKVVK-a2-b6 | Cβ | Cβ | 83 | 78 | 0 | 34.16 |
| KMYESFIESLPFLK-RVKGR-a1-b3 | RIIβ | Cβ | 263 | 192 | 0.37 | 33.91 |
| LVKDGEHVIDQGDDGDNFYVIDR-RIIVKNNAK-a3-b5 | RIIβ | RIIβ | 174 | 256 | 0 | 33.79 |
| NGVSDIKTHK-KVEAPFIPK-a7-b1 | Cβ | Cβ | 292 | 309 | 0.81 | 33.79 |
| KGSEVESVKEFLAK-AKEDFLR-a9-b2 | Cβ | Cβ | 16 | 23 | 0.77 | 33.42 |
| LKQIEHTLNEK-QKVVK-a2-b2 | Cβ | Cβ | 83 | 78 | 0.77 | 33.38 |
| QIEHTLNEKR-AKEDFLR-a9-b2 | Cβ | Cβ | 92 | 23 | 0 | 33.34 |
| LKVVDVIGTK-VVKLK-a2-b3 | RIIβ | Cβ | 285 | 81 | 0.38 | 33.24 |
| RKGKSEVEENGAVEIAR-ITMKR-a4-b4 | RIIβ | RIIβ | 328 | 324 | 0.33 | 33.1 |
| RKGKSEVEENGAVEIAR-ITMKR-a2-b4 | RIIβ | RIIβ | 326 | 324 | 0.31 | 32.73 |
| HKATEQYYAMKILDK-VVKLK-a11-b3 | Cβ | Cβ | 72 | 81 | 0.57 | 32.69 |
| HKATEQYYAMK-KKTLGTGSFGR-a2-b2 | Cβ | Cβ | 63 | 47 | 0.52 | 32.69 |
| HKATEQYYAMKILDKQK-AKEDFLR-a11-b2 | Cβ | Cβ | 72 | 23 | 0.74 | 32.64 |
| LVKDGEHVIDQGDDGDNFYVIDR-IIHPKTDDQR-a3-b5 | RIIβ | RIIβ | 174 | 135 | 0.69 | 32.57 |
| KWENPPPSNAGLEDFER-KVEAPFIPK-a1-b1 | Cβ | Cβ | 29 | 309 | 0 | 31.72 |
| ATEQYYAMKILDK-LKQIEHTLNEKR-a9-b2 | Cβ | Cβ | 72 | 83 | 0.37 | 31.56 |
| MYESFIESLPFLKSLEFSER-KGTAR-a13-b1 | RIIβ | RIIβ | 276 | 46 | 0 | 31.48 |
| KGKSEVEENGAVEIAR-NNAKK-a3-b4 | RIIβ | RIIβ | 328 | 260 | 0.56 | 31.08 |
| ATEQYYAMKILDK-LKQIEHTLNEKR-a9-b11 | Cβ | Cβ | 72 | 92 | 0.21 | 31.06 |
| FPSHFSSDLKDLLR-IVSGKVR-a10-b5 | Cβ | Cβ | 266 | 254 | 0.73 | 30.91 |
| KMYESFIESLPFLK-NNAKKR-a1-b5 | RIIβ | RIIβ | 263 | 261 | 0.92 | 30.9 |
| KWENPPPSNAGLEDFER-NGVSDIKTHK-a1-b7 | Cβ | Cβ | 29 | 292 | 0 | 30.77 |
| AKEDFLR-RVKGR-a2-b3 | Cβ | Cβ | 23 | 192 | 0 | 30.56 |
| KVEAPFIPK-AKEDFLR-a1-b2 | Cβ | Cβ | 309 | 23 | 0 | 30.51 |
| ATEQYYAMKILDK-LKQIEHTLNEK-a9-b2 | Cβ | Cβ | 72 | 83 | 0.36 | 29.82 |
| ATEQYYAMKILDK-QIEHTLNEKR-a9-b9 | Cβ | Cβ | 72 | 92 | 0 | 29.68 |
| AKEDFLRK-RVKGR-a2-b3 | Cβ | Cβ | 23 | 192 | 0.51 | 29.63 |
| ATEQYYAMKILDK-RVKGR-a9-b3 | Cβ | Cβ | 72 | 192 | 0.6 | 29.39 |
| KWENPPPSNAGLEDFER-AKEDFLR-a1-b2 | Cβ | Cβ | 29 | 23 | 0.9 | 29.07 |
| FGNLKNGVSDIKTHK-KGSEVESVKEFLAK-a12-b9 | Cβ | Cβ | 292 | 16 | 0.82 | 28.68 |
| KMYESFIESLPFLK-LKVVDVIGTK-a1-b2 | RIIβ | RIIβ | 263 | 285 | 0 | 28.64 |
| DLKPENLLIDHQGYIQVTDFGFAKR-AKEDFLR-a24-b2 | Cβ | Cβ | 189 | 23 | 0.37 | 28.33 |
| GQYFGELALVTNKPRAASAHAIGTVK-LKQIEHTLNEKR-a13-b11 | RIIβ | Cβ | 357 | 92 | 0.21 | 28.11 |
| KMYESFIESLPFLKSLEFSER-KGTAR-a14-b1 | RIIβ | RIIβ | 276 | 46 | 0.3 | 27.76 |
| LKQIEHTLNEKR-RVKGR-a2-b3 | Cβ | Cβ | 83 | 192 | 0 | 27.69 |
| KWENPPPSNAGLEDFER-IVSGKVR-a1-b5 | Cβ | Cβ | 29 | 254 | 0 | 27.65 |
| QIEHTLNEKR-VVKLK-a9-b3 | Cβ | Cβ | 92 | 81 | 0.32 | 27.61 |
| LKQIEHTLNEKR-ILDKQKVVK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.88 | 27.54 |
| RFGNLKNGVSDIKTHK-AKEDFLR-a13-b2 | Cβ | Cβ | 292 | 23 | 0.58 | 27.51 |
| LEYSFKDNSNLYMVMEYVPGGEMFSHLR-ILDKQK-a6-b4 | Cβ | Cβ | 111 | 76 | 0 | 27.34 |
| KWENPPPSNAGLEDFER-ATEQYYAMKILDKQK-a1-b13 | Cβ | Cβ | 29 | 76 | 0.35 | 26.82 |
| KWENPPPSNAGLEDFER-LKQIEHTLNEKR-a1-b11 | Cβ | Cβ | 29 | 92 | 0 | 26.52 |
| HKATEQYYAMKILDK-LKQIEHTLNEKR-a11-b11 | Cβ | Cβ | 72 | 92 | 0.36 | 26.39 |
| KMYESFIESLPFLK-RIIVKNNAK-a1-b5 | RIIβ | RIIβ | 263 | 256 | 0.87 | 25.21 |
| ATEQYYAMKILDKQK-LKQIEHTLNEKR-a9-b2 | Cβ | Cβ | 72 | 83 | 0.69 | 25.18 |
| KWENPPPSNAGLEDFER-LKQIEHTLNEKR-a1-b2 | Cβ | Cβ | 29 | 83 | 0 | 25.17 |
| FGNLKNGVSDIKTHK-NLLQVDLTKR-a5-b9 | Cβ | Cβ | 285 | 279 | 0.38 | 25.16 |
| FPSHFSSDLKDLLRNLLQVDLTKR-IVSGKVR-a10-b5 | Cβ | Cβ | 266 | 254 | 0.56 | 25.13 |
| GQYFGELALVTNKPR-FGNLKNGVSDIK-a13-b5 | RIIβ | Cβ | 357 | 285 | 0.33 | 24.94 |
| NLLQVDLTKRFGNLK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 279 | 292 | 0.91 | 24.68 |
| GQYFGELALVTNKPRAASAHAIGTVK-LKQIEHTLNEKR-a13-b2 | RIIβ | Cβ | 357 | 83 | 0.17 | 24.65 |
| LKQIEHTLNEK-RVKGR-a2-b3 | Cβ | Cβ | 83 | 192 | 0.49 | 24.35 |
| ATEQYYAMKILDK-QKVVKLK-a9-b5 | Cβ | Cβ | 72 | 81 | 0.9 | 23.59 |
| KWENPPPSNAGLEDFER-QIEHTLNEKR-a1-b9 | Cβ | Cβ | 29 | 92 | 0.35 | 23.45 |
| ATEQYYAMKILDKQK-NGVSDIKTHK-a13-b7 | Cβ | Cβ | 76 | 292 | 0.93 | 22.86 |
| HKATEQYYAMKILDK-QIEHTLNEKR-a11-b9 | Cβ | Cβ | 72 | 92 | 0.4 | 22.67 |
| LKQIEHTLNEK-LKVVDVIGTK-a2-b2 | Cβ | RIIβ | 83 | 285 | 0.5 | 22.52 |
| RKMYESFIESLPFLKSLEFSER-KGTAR-a15-b1 | RIIβ | RIIβ | 276 | 46 | 0 | 22.27 |
| HKATEQYYAMKILDKQK-QIEHTLNEKR-a11-b9 | Cβ | Cβ | 72 | 92 | 0.76 | 22.07 |
| HKATEQYYAMKILDKQK-LKQIEHTLNEKR-a11-b2 | Cβ | Cβ | 72 | 83 | 0.71 | 21.62 |
| RKMYESFIESLPFLKSLEFSER-IIVKNNAK-a15-b4 | RIIβ | RIIβ | 276 | 256 | 0.82 | 21.54 |
| NLDPEQMSQVLDAMFEKLVK-IIHPKTDDQR-a17-b5 | RIIβ | RIIβ | 171 | 135 | 0.53 | 21.33 |
| LKQIEHTLNEKR-AKEDFLR-a11-b2 | Cβ | Cβ | 92 | 23 | 0 | 21.26 |
| ATEQYYAMKILDKQK-VVKLK-a9-b3 | Cβ | Cβ | 72 | 81 | 0.81 | 20.99 |
| LKQIEHTLNEKR-HKATEQYYAMK-a2-b2 | Cβ | Cβ | 83 | 63 | 0 | 20.88 |
| NGVSDIKTHK-QIEHTLNEKR-a7-b9 | Cβ | Cβ | 292 | 92 | 0 | 20.68 |
| QIEHTLNEKR-ILDKQKVVK-a9-b4 | Cβ | Cβ | 92 | 76 | 0.91 | 20.34 |
| KWENPPPSNAGLEDFER-KKTLGTGSFGR-a1-b2 | Cβ | Cβ | 29 | 47 | 0.36 | 20.14 |
The first column lists the amino acid sequence of cross-linked peptides and the positions of the cross-linked lysine residues. DeltaS is a measure for how close the best assigned hit was scored in regard to the second best. The peptides are listed in order of linear discriminant score, which is a weighted sum of the difference used to assess the quality of the composite MS2 spectrum as calculated by xQuest.
Table S3.
| Cross-linked peptide sequence | Protein 1 | Protein 2 | Position 1 | Position 2 | deltaS | Linear discriminant score |
|---|---|---|---|---|---|---|
| LKQIEHTLNEK-ILDKQK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.52 | 50.16 |
| GKSEVEENGAVEIAR-GQYFGELALVTNKPR-a2-b13 | RIIβ | RIIβ | 328 | 357 | 0 | 45.44 |
| RKGKSEVEENGAVEIAR-GQYFGELALVTNKPR-a2-b13 | RIIβ | RIIβ | 326 | 357 | 0 | 44.79 |
| KMYESFIESLPFLK-NNAKKR-a1-b4 | RIIβ | RIIβ | 263 | 260 | 0.4 | 44.5 |
| IIHPKTDDQR-LKVVDVIGTK-a5-b2 | RIIβ | RIIβ | 135 | 285 | 0 | 44.42 |
| FPSHFSSDLKDLLR-KGTAR-a10-b1 | Cβ | RIIβ | 266 | 46 | 0.48 | 44.41 |
| NLLQVDLTKR-IVSGKVR-a9-b5 | Cβ | Cβ | 279 | 254 | 0.63 | 42.61 |
| KMYESFIESLPFLK-IIVKNNAK-a1-b4 | RIIβ | RIIβ | 263 | 256 | 0 | 42.35 |
| HKATEQYYAMKILDKQK-VVKLK-a11-b3 | Cβ | Cβ | 72 | 81 | 0.76 | 42.24 |
| NGVSDIKTHK-AKEDFLR-a7-b2 | Cβ | Cβ | 292 | 23 | 0 | 41.27 |
| KGKSEVEENGAVEIAR-ITMKR-a1-b4 | RIIβ | RIIβ | 326 | 324 | 0.94 | 41.25 |
| ILDKQK-VVKLK-a4-b3 | Cβ | Cβ | 76 | 81 | 0.88 | 41.17 |
| KGKSEVEENGAVEIAR-ITMKR-a3-b4 | RIIβ | RIIβ | 328 | 324 | 0.94 | 41.05 |
| KGSEVESVKEFLAK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 16 | 292 | 0.57 | 40.47 |
| KKTLGTGSFGR-VEAPFIPKFR-a2-b8 | Cβ | Cβ | 47 | 317 | 0.92 | 40.23 |
| LKQIEHTLNEKR-ILDKQK-a11-b4 | Cβ | Cβ | 92 | 76 | 0 | 40.07 |
| LKQIEHTLNEK-AKEDFLR-a2-b2 | Cβ | Cβ | 83 | 23 | 0.6 | 38.43 |
| KKTLGTGSFGR-VMLVKHK-a2-b5 | Cβ | Cβ | 47 | 61 | 0 | 38.36 |
| AKEDFLRK-RVKGR-a2-b3 | Cβ | Cβ | 23 | 192 | 0.52 | 37.98 |
| LKVVDVIGTK-KGTAR-a2-b1 | RIIβ | RIIβ | 285 | 46 | 0 | 37.96 |
| LVKDGEHVIDQGDDGDNFYVIDR-KGTAR-a3-b1 | RIIβ | RIIβ | 174 | 46 | 0 | 37.89 |
| ATEQYYAMKILDK-VMLVKHK-a9-b5 | Cβ | Cβ | 72 | 61 | 0.56 | 37.59 |
| LKQIEHTLNEKR-AKEDFLR-a11-b2 | Cβ | Cβ | 92 | 23 | 0 | 37.04 |
| LVKDGEHVIDQGDDGDNFYVIDR-IIVKNNAK-a3-b4 | RIIβ | RIIβ | 174 | 256 | 0.22 | 36.73 |
| KMYESFIESLPFLK-KGTAR-a1-b1 | RIIβ | RIIβ | 263 | 46 | 0 | 36.55 |
| LKQIEHTLNEKR-ILDKQKVVK-a2-b6 | Cβ | Cβ | 83 | 78 | 0.88 | 36.5 |
| KVEAPFIPKFR-AKEDFLR-a9-b2 | Cβ | Cβ | 317 | 23 | 0.36 | 36.29 |
| IIHPKTDDQR-KGTAR-a5-b1 | RIIβ | RIIβ | 135 | 46 | 0.46 | 35.89 |
| LKVVDVIGTK-NNAKK-a2-b4 | RIIβ | RIIβ | 285 | 260 | 0 | 35.82 |
| HKATEQYYAMKILDK-LKQIEHTLNEKR-a11-b2 | Cβ | Cβ | 72 | 83 | 0.43 | 35.37 |
| LVKDGEHVIDQGDDGDNFYVIDR-IVSGKVR-a3-b5 | RIIβ | Cβ | 174 | 254 | 0.17 | 34.9 |
| LKVVDVIGTK-IIVKNNAK-a2-b4 | RIIβ | RIIβ | 285 | 256 | 0 | 34.62 |
| HKATEQYYAMKILDK-LKQIEHTLNEKR-a11-b11 | Cβ | Cβ | 72 | 92 | 0.32 | 34.59 |
| HKATEQYYAMKILDKQK-AKEDFLR-a11-b2 | Cβ | Cβ | 72 | 23 | 0.68 | 34.29 |
| HKATEQYYAMK-AKEDFLR-a2-b2 | Cβ | Cβ | 63 | 23 | 0 | 33.97 |
| KGSEVESVKEFLAK-AKEDFLR-a9-b2 | Cβ | Cβ | 16 | 23 | 0.7 | 33.9 |
| LVKDGEHVIDQGDDGDNFYVIDR-IIHPKTDDQR-a3-b5 | RIIβ | RIIβ | 174 | 135 | 0 | 33.36 |
| AKEDFLR-VMLVKHK-a2-b5 | Cβ | Cβ | 23 | 61 | 0 | 33.36 |
| IIHPKTDDQR-IIVKNNAK-a5-b4 | RIIβ | RIIβ | 135 | 256 | 0 | 33.19 |
| HKATEQYYAMK-ILDKQK-a2-b4 | Cβ | Cβ | 63 | 76 | 0 | 32.97 |
| VMLVKHK-ILDKQK-a5-b4 | Cβ | Cβ | 61 | 76 | 0 | 32.54 |
| GQYFGELALVTNKPR-KGTAR-a13-b1 | RIIβ | RIIβ | 357 | 46 | 0 | 32.41 |
| KKTLGTGSFGR-KVEAPFIPKFR-a1-b9 | Cβ | Cβ | 46 | 317 | 0.79 | 32.41 |
| NGVSDIKTHK-KVEAPFIPK-a7-b1 | Cβ | Cβ | 292 | 309 | 0 | 32.36 |
| KWENPPPSNAGLEDFER-NGVSDIKTHK-a1-b7 | Cβ | Cβ | 29 | 292 | 0.31 | 32.19 |
| KWENPPPSNAGLEDFER-ILDKQK-a1-b4 | Cβ | Cβ | 29 | 76 | 0 | 32.07 |
| DLKPENLLIDHQGYIQVTDFGFAKR-AKEDFLR-a24-b2 | Cβ | Cβ | 189 | 23 | 0.29 | 32.03 |
| FGNLKNGVSDIK-AKEDFLR-a5-b2 | Cβ | Cβ | 285 | 23 | 0 | 31.84 |
| LKQIEHTLNEKR-ILDKQKVVK-a11-b6 | Cβ | Cβ | 92 | 78 | 0.75 | 31.51 |
| LKVVDVIGTK-VVKLK-a2-b3 | RIIβ | Cβ | 285 | 81 | 0 | 31.35 |
| KWENPPPSNAGLEDFER-KVEAPFIPK-a1-b1 | Cβ | Cβ | 29 | 309 | 0.42 | 30.72 |
| LKQIEHTLNEKR-HKATEQYYAMK-a2-b2 | Cβ | Cβ | 83 | 63 | 0.4 | 30.52 |
| QIEHTLNEKR-VVKLK-a9-b3 | Cβ | Cβ | 92 | 81 | 0 | 29.73 |
| KVEAPFIPK-AKEDFLR-a1-b2 | Cβ | Cβ | 309 | 23 | 0 | 29.51 |
| LKQIEHTLNEKR-HKATEQYYAMK-a11-b2 | Cβ | Cβ | 92 | 63 | 0 | 29.29 |
| LKVVDVIGTK-ITMKR-a2-b4 | RIIβ | RIIβ | 285 | 324 | 0 | 28.93 |
| NLLQVDLTKRFGNLK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 279 | 292 | 0.91 | 27.85 |
| KMYESFIESLPFLKSLEFSER-KGTAR-a14-b1 | RIIβ | RIIβ | 276 | 46 | 0.36 | 27.07 |
| NGVSDIKTHK-EFLAKAK-a7-b5 | Cβ | Cβ | 292 | 21 | 0.72 | 26.75 |
| KWENPPPSNAGLEDFER-LKQIEHTLNEKR-a1-b11 | Cβ | Cβ | 29 | 92 | 0.28 | 26.43 |
| NGVSDIKTHK-QIEHTLNEKR-a7-b9 | Cβ | Cβ | 292 | 92 | 0.7 | 26.17 |
| HKATEQYYAMKILDKQK-KWENPPPSNAGLEDFER-a11-b1 | Cβ | Cβ | 72 | 29 | 0.51 | 25.99 |
| KMYESFIESLPFLK-LKVVDVIGTK-a1-b2 | RIIβ | RIIβ | 263 | 285 | 0.48 | 25.77 |
| FGNLKNGVSDIK-ICEKDR-a5-b4 | Cβ | AKAP18α | 285 | 19 | 0.86 | 24.44 |
| ATEQYYAMKILDKQK-NGVSDIKTHK-a13-b7 | Cβ | Cβ | 76 | 292 | 0.94 | 24.39 |
| QIEHTLNEKR-VMLVKHK-a9-b5 | Cβ | Cβ | 92 | 61 | 0 | 24 |
| KWENPPPSNAGLEDFER-LKQIEHTLNEKR-a1-b2 | Cβ | Cβ | 29 | 83 | 0 | 23.73 |
| FGNLKNGVSDIKTHK-KGSEVESVKEFLAK-a5-b9 | Cβ | Cβ | 285 | 16 | 0.54 | 23.66 |
| KGSEVESVKEFLAKAK-ILDKQK-a9-b4 | Cβ | Cβ | 16 | 76 | 0.89 | 22.35 |
| KWENPPPSNAGLEDFER-HKATEQYYAMK-a1-b2 | Cβ | Cβ | 29 | 63 | 0.24 | 22.01 |
| ATEQYYAMKILDKQK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 72 | 292 | 0.38 | 21.23 |
| AVQQYLEETQNKKQPGEGNSTKAEEGDR-KMYESFIESLPFLK-a22-b1 | AKAP18α | RIIβ | 65 | 263 | 0 | 21.07 |
| ATEQYYAMKILDK-EFLAKAK-a9-b5 | Cβ | Cβ | 72 | 21 | 0 | 20.79 |
| FGNLKNGVSDIKTHK-NLLQVDLTKR-a5-b9 | Cβ | Cβ | 285 | 279 | 0.65 | 20.68 |
| KTLGTGSFGRVMLVK-ILDKQK-a1-b4 | Cβ | Cβ | 47 | 76 | 0.77 | 20.65 |
The first column lists the amino acid sequence of cross-linked peptides and the positions of the cross-linked lysine residues. DeltaS is a measure for how close the best assigned hit was scored in regard to the second best. The peptides are listed in order of linear discriminant score, which is a weighted sum of the difference used to assess the quality of the composite MS2 spectrum as calculated by xQuest.
Nine different types of cross-link were identified linking RIIβ K46 at the C terminus of the D/D domain to lysines visible in crystal structures of RIIβ–C (14, 37). In six cases, RIIβ K46 is coupled to sites in CNBB (positions 263, 266, 276, 326, 328, and 357), with another partner lysine (Cβ K285) that projects over CNBB (Fig. 4C). This pattern of cross-linking is consistent with the D/D domain lying between the CNBB domains of the RIIβ dimer. XL-MS with purified RIIβ–Cβ–AKAP18α led to identification of two interlinks involving AKAP18α (Table S3): AKAP18α K19 linked to Cβ K285 and AKAP18α K65 linked to RIIβ K263. AKAP18α K19 is between the N-terminal lipid attachment sites and the R-subunit anchoring helix of the AKAP (positions 27–42), whereas K65 is near to the C terminus of the anchoring protein. These interlinks are consistent with AKAP18α docking to a D/D located between the CNBB domains of the RII dimer (Fig. 4C). We also analyzed a complex of RIIα and Cβ using XL-MS (Fig. S4A and Table S4). This analysis was less powerful, as the only reference lysine within the first 100 amino acids of RIIα (K71) is midway between the autoinhibitory sequence and D/D domain (Fig. S4B). Furthermore, there are no crystal structures of tetrameric RIIα–C for reference. Nevertheless, the pattern of interlinks between RIIα K71 and Cβ (Fig. S4C) is consistent with the D/D adopting a similar position relative to the C subunit in both RII isoforms.
Fig. S4.
Table S4.
| Cross-linked peptide sequence | Protein 1 | Protein 2 | Position 1 | Position 2 | deltaS | Linear discriminant score |
|---|---|---|---|---|---|---|
| TKSNKDGGNQEVEIAR-SNKDGGNQEVEIAR-a2-b3 | RIIα | RIIα | 311 | 314 | 0.75 | 54.11 |
| KMFESFIESVPLLK-MKIVDVIGEK-a1-b2 | RIIα | RIIα | 246 | 268 | 0 | 49.14 |
| NISHYEEQLVKMFGSSVDLGNLGQ-TKSNKDGGNQEVEIAR-a11-b5 | RIIα | RIIα | 387 | 314 | 0 | 48.79 |
| NISHYEEQLVKMFGSSVDLGNLGQ-TKSNKDGGNQEVEIAR-a11-b2 | RIIα | RIIα | 387 | 311 | 0 | 47.93 |
| KVEAPFIPK-AKEDFLR-a1-b2 | Cβ | Cβ | 309 | 23 | 0 | 47.4 |
| SNKDGGNQEVEIAR-SNKDGGNQEVEIAR-a3-b3 | RIIα | RIIα | 314 | 314 | 0 | 45.9 |
| NLLQVDLTKR-IVSGKVR-a9-b5 | Cβ | Cβ | 279 | 254 | 0.42 | 45.65 |
| TKSNKDGGNQEVEIAR-SNKDGGNQEVEIAR-a5-b3 | RIIα | RIIα | 314 | 314 | 0.88 | 44.6 |
| NISHYEEQLVKMFGSSVDLGNLGQ-SNKDGGNQEVEIAR-a11-b3 | RIIα | RIIα | 387 | 314 | 0 | 43.7 |
| MKIVDVIGEK-ILDKQK-a2-b4 | RIIα | Cβ | 268 | 76 | 0 | 43.25 |
| MKIVDVIGEK-IYKDGER-a2-b3 | RIIα | RIIα | 268 | 279 | 0 | 42.83 |
| TKSNKDGGNQEVEIAR-TKSNKDGGNQEVEIAR-a2-b2 | RIIα | RIIα | 311 | 311 | 0.79 | 42.79 |
| TKSNKDGGNQEVEIAR-VIHPKTDEQR-a5-b5 | RIIα | RIIα | 314 | 118 | 0.4 | 41.46 |
| TKSNKDGGNQEVEIAR-TKSNKDGGNQEVEIAR-a5-b5 | RIIα | RIIα | 314 | 314 | 0.8 | 41.38 |
| SNKDGGNQEVEIAR-IYKDGER-a3-b3 | RIIα | RIIα | 314 | 279 | 0 | 41.36 |
| LKQIEHTLNEKR-ILDKQK-a11-b4 | Cβ | Cβ | 92 | 76 | 0 | 41.14 |
| KMFESFIESVPLLK-IIVKNNAK-a1-b4 | RIIα | RIIα | 246 | 239 | 0 | 41.05 |
| FGNLKNGVSDIKTHK-AKEDFLR-a12-b2 | Cβ | Cβ | 292 | 23 | 0 | 40.92 |
| VIHPKTDEQR-RIIVKNNAK-a5-b5 | RIIα | RIIα | 118 | 239 | 0 | 40.91 |
| HKATEQYYAMK-KKTLGTGSFGR-a2-b2 | Cβ | Cβ | 63 | 47 | 0 | 40.69 |
| LKQIEHTLNEK-ILDKQKVVK-a2-b6 | Cβ | Cβ | 83 | 78 | 0.94 | 40.49 |
| LKQIEHTLNEK-ILDKQK-a2-b4 | Cβ | Cβ | 83 | 76 | 0 | 40.14 |
| LKQIEHTLNEKR-MKIVDVIGEK-a2-b2 | Cβ | RIIα | 83 | 268 | 0 | 40.12 |
| GTYDILVTKDNQTR-MKIVDVIGEKIYK-a9-b2 | RIIα | RIIα | 186 | 268 | 0.59 | 39.88 |
| GTYDILVTKDNQTR-MKIVDVIGEK-a9-b2 | RIIα | RIIα | 186 | 268 | 0.57 | 39.83 |
| RNISHYEEQLVKMFGSSVDLGNLGQ-TKSNKDGGNQEVEIAR-a12-b5 | RIIα | RIIα | 387 | 314 | 0.5 | 39.16 |
| RKMFESFIESVPLLK-IIVKNNAK-a2-b4 | RIIα | RIIα | 246 | 239 | 0.82 | 39.06 |
| VIHPKTDEQR-IIVKNNAK-a5-b4 | RIIα | RIIα | 118 | 239 | 0 | 38.1 |
| KKTLGTGSFGR-VEAPFIPKFR-a2-b8 | Cβ | Cβ | 47 | 317 | 0.79 | 37.97 |
| TKSNKDGGNQEVEIAR-VIHPKTDEQR-a2-b5 | RIIα | RIIα | 311 | 118 | 0.4 | 37.5 |
| KVEAPFIPKFR-VMLVKHK-a9-b5 | Cβ | Cβ | 317 | 61 | 0 | 37.29 |
| AKEDFLR-VMLVKHK-a2-b5 | Cβ | Cβ | 23 | 61 | 0 | 37.08 |
| KKTLGTGSFGR-VMLVKHK-a2-b5 | Cβ | Cβ | 47 | 61 | 0 | 36.98 |
| DLKPENLLIDHQGYIQVTDFGFAKR-AKEDFLR-a24-b2 | Cβ | Cβ | 189 | 23 | 0.36 | 36.83 |
| LKQIEHTLNEKR-ILDKQKVVK-a2-b6 | Cβ | Cβ | 83 | 78 | 0.67 | 36.64 |
| KMFESFIESVPLLK-NNAKKR-a1-b4 | RIIα | RIIα | 246 | 243 | 0 | 36.54 |
| TKSNKDGGNQEVEIAR-IYKDGER-a2-b3 | RIIα | RIIα | 311 | 279 | 0.22 | 36.37 |
| LKQIEHTLNEKR-ILDKQK-a2-b4 | Cβ | Cβ | 83 | 76 | 0 | 36.2 |
| NGVSDIKTHK-AKEDFLR-a7-b2 | Cβ | Cβ | 292 | 23 | 0 | 36.01 |
| THKWFATTDWIAIYQR-FGNLKNGVSDIK-a3-b5 | Cβ | Cβ | 295 | 285 | 0 | 35.7 |
| NGVSDIKTHK-AKEDFLRK-a7-b2 | Cβ | Cβ | 292 | 23 | 0.64 | 35.66 |
| LKQIEHTLNEKR-ILDKQKVVK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.73 | 35.37 |
| VIHPKTDEQR-IYKDGER-a5-b3 | RIIα | RIIα | 118 | 279 | 0 | 35.32 |
| GTYDILVTKDNQTR-GTYDILVTKDNQTR-a9-b9 | RIIα | RIIα | 186 | 186 | 0.82 | 35.11 |
| RKMFESFIESVPLLK-IIVKNNAKK-a2-b8 | RIIα | RIIα | 246 | 243 | 0.47 | 35 |
| TKSNKDGGNQEVEIAR-GTYDILVTKDNQTR-a5-b9 | RIIα | RIIα | 314 | 186 | 0.69 | 34.86 |
| IVKADEHVIDQGDDGDNFYVIER-MKIVDVIGEK-a3-b2 | RIIα | RIIα | 157 | 268 | 0 | 34.71 |
| SNKDGGNQEVEIAR-IIVKNNAK-a3-b4 | RIIα | RIIα | 314 | 239 | 0 | 34.7 |
| VIHPKTDEQR-ILDKQK-a5-b4 | RIIα | Cβ | 118 | 76 | 0 | 34.68 |
| TKSNKDGGNQEVEIAR-MKIVDVIGEK-a2-b2 | RIIα | RIIα | 311 | 268 | 0.44 | 34.62 |
| GTYDILVTKDNQTR-VIHPKTDEQR-a9-b5 | RIIα | RIIα | 186 | 118 | 0.5 | 34.53 |
| IVKADEHVIDQGDDGDNFYVIER-VIHPKTDEQR-a3-b5 | RIIα | RIIα | 157 | 118 | 0.41 | 34.48 |
| FPSHFSSDLKDLLR-IVSGKVR-a10-b5 | Cβ | Cβ | 266 | 254 | 0 | 34.44 |
| GTYDILVTKDNQTR-SNKDGGNQEVEIAR-a9-b3 | RIIα | RIIα | 186 | 314 | 0.62 | 34.29 |
| SNKDGGNQEVEIAR-NNAKKR-a3-b4 | RIIα | RIIα | 314 | 243 | 0 | 34.29 |
| KKTLGTGSFGR-KVEAPFIPKFR-a2-b9 | Cβ | Cβ | 47 | 317 | 0 | 34.05 |
| KKTLGTGSFGR-VMLVKHK-a1-b5 | Cβ | Cβ | 46 | 61 | 0 | 33.96 |
| NGVSDIKTHKWFATTDWIAIYQR-AKEDFLR-a10-b2 | Cβ | Cβ | 295 | 23 | 0 | 33.93 |
| GTYDILVTKDNQTR-IVSGKVR-a9-b5 | RIIα | Cβ | 186 | 254 | 0.54 | 33.77 |
| TKSNKDGGNQEVEIAR-MKIVDVIGEK-a5-b2 | RIIα | RIIα | 314 | 268 | 0.44 | 33.75 |
| FGNLKNGVSDIK-NLLQVDLTKR-a5-b9 | Cβ | Cβ | 285 | 279 | 0 | 33.45 |
| TKSNKDGGNQEVEIAR-IYKDGER-a5-b3 | RIIα | RIIα | 314 | 279 | 0.34 | 33.43 |
| NGVSDIKTHKWFATTDWIAIYQR-AKEDFLR-a7-b2 | Cβ | Cβ | 292 | 23 | 0 | 33.38 |
| MKIVDVIGEK-AKEDFLR-a2-b2 | RIIα | Cβ | 268 | 23 | 0 | 33.33 |
| DILLFKNLDQEQLSQVLDAMFER-IYKDGER-a6-b3 | RIIα | RIIα | 137 | 279 | 0 | 33.32 |
| IVDVIGEKIYKDGER-GTYDILVTKDNQTR-a8-b9 | RIIα | RIIα | 276 | 186 | 0.64 | 33.26 |
| LKQIEHTLNEKR-AKEDFLR-a2-b2 | Cβ | Cβ | 83 | 23 | 0 | 33.21 |
| SNKDGGNQEVEIAR-VIHPKTDEQR-a3-b5 | RIIα | RIIα | 314 | 118 | 0 | 33.18 |
| HKATEQYYAMKILDK-LKQIEHTLNEKR-a11-b2 | Cβ | Cβ | 72 | 83 | 0.66 | 33.12 |
| TKSNKDGGNQEVEIAR-GQYFGELALVTNKPR-a5-b13 | RIIα | RIIα | 314 | 341 | 0.79 | 33.05 |
| TKSNKDGGNQEVEIAR-IIVKNNAK-a2-b4 | RIIα | RIIα | 311 | 239 | 0 | 32.91 |
| FGNLKNGVSDIKTHK-AKEDFLRK-a12-b2 | Cβ | Cβ | 292 | 23 | 0.37 | 32.78 |
| VADAKGDSESEEDEDLEVPVPSR-NLLQVDLTKR-a5-b9 | RIIα | Cβ | 71 | 279 | 0 | 32.74 |
| RNISHYEEQLVKMFGSSVDLGNLGQ-TKSNKDGGNQEVEIAR-a12-b2 | RIIα | RIIα | 387 | 311 | 0.65 | 32.55 |
| KVEAPFIPK-ILDKQK-a1-b4 | Cβ | Cβ | 309 | 76 | 0 | 32.38 |
| KWENPPPSNAGLEDFER-AKEDFLR-a1-b2 | Cβ | Cβ | 29 | 23 | 0.24 | 32.34 |
| KTLGTGSFGR-VMLVKHK-a1-b5 | Cβ | Cβ | 47 | 61 | 0 | 32.16 |
| FPSHFSSDLKDLLR-NLLQVDLTKR-a10-b9 | Cβ | Cβ | 266 | 279 | 0.81 | 32.16 |
| VVKLKQIEHTLNEK-ILDKQK-a3-b4 | Cβ | Cβ | 81 | 76 | 0.95 | 32.11 |
| HKATEQYYAMK-KKTLGTGSFGR-a2-b1 | Cβ | Cβ | 63 | 46 | 0.55 | 32.07 |
| ATEQYYAMKILDK-VMLVKHK-a9-b5 | Cβ | Cβ | 72 | 61 | 0 | 31.86 |
| IVKADEHVIDQGDDGDNFYVIER-GTYDILVTKDNQTR-a3-b9 | RIIα | RIIα | 157 | 186 | 0.54 | 31.84 |
| QIEHTLNEKR-ILDKQK-a9-b4 | Cβ | Cβ | 92 | 76 | 0.79 | 31.8 |
| RKMFESFIESVPLLK-IIVKNNAKK-a2-b4 | RIIα | RIIα | 246 | 239 | 0 | 31.66 |
| TKSNKDGGNQEVEIAR-GQYFGELALVTNKPR-a2-b13 | RIIα | RIIα | 311 | 341 | 0 | 31.65 |
| GQYFGELALVTNKPR-SNKDGGNQEVEIAR-a13-b3 | RIIα | RIIα | 341 | 314 | 0 | 31.54 |
| NGVSDIKTHK-KVEAPFIPK-a7-b1 | Cβ | Cβ | 292 | 309 | 0 | 31.26 |
| FGNLKNGVSDIKTHK-NLLQVDLTKR-a5-b9 | Cβ | Cβ | 285 | 279 | 0.79 | 31.19 |
| QSLGHPPPEPGPDRVADAKGDSESEEDEDLEVPVPSR-GTYDILVTKDNQTR-a19-b9 | RIIα | RIIα | 71 | 186 | 0.67 | 31.01 |
| TKSNKDGGNQEVEIAR-GTYDILVTKDNQTR-a2-b9 | RIIα | RIIα | 311 | 186 | 0.77 | 30.84 |
| QIEHTLNEKR-AKEDFLR-a9-b2 | Cβ | Cβ | 92 | 23 | 0 | 30.7 |
| LKQIEHTLNEKR-ILDKQKVVK-a11-b6 | Cβ | Cβ | 92 | 78 | 0.81 | 30.53 |
| KVEAPFIPKFR-AKEDFLR-a9-b2 | Cβ | Cβ | 317 | 23 | 0.58 | 30.17 |
| KTLGTGSFGR-VEAPFIPKFR-a1-b8 | Cβ | Cβ | 47 | 317 | 0 | 30.16 |
| LKQIEHTLNEKR-IIVKNNAK-a2-b4 | Cβ | RIIα | 83 | 239 | 0.58 | 30.08 |
| GTYDILVTKDNQTR-ILDKQKVVK-a9-b6 | RIIα | Cβ | 186 | 78 | 0.93 | 29.97 |
| TKSNKDGGNQEVEIAR-NNAKKR-a2-b4 | RIIα | RIIα | 311 | 243 | 0.52 | 29.59 |
| VADAKGDSESEEDEDLEVPVPSR-IVSGKVR-a5-b5 | RIIα | Cβ | 71 | 254 | 0 | 29.55 |
| SNKDGGNQEVEIAR-MKIVDVIGEK-a3-b2 | RIIα | RIIα | 314 | 268 | 0 | 29.5 |
| VIHPKTDEQR-ILDKQKVVK-a5-b4 | RIIα | Cβ | 118 | 76 | 0 | 29.25 |
| IVDVIGEKIYKDGER-IIVKNNAK-a8-b4 | RIIα | RIIα | 276 | 239 | 0 | 29.15 |
| MKIVDVIGEK-IIVKNNAK-a2-b4 | RIIα | RIIα | 268 | 239 | 0 | 28.97 |
| LKQIEHTLNEKR-ILDKQKVVK-a11-b4 | Cβ | Cβ | 92 | 76 | 0 | 28.66 |
| LEYSFKDNSNLYMVMEYVPGGEMFSHLR-QIEHTLNEKR-a6-b9 | Cβ | Cβ | 111 | 92 | 0.22 | 28.59 |
| ATEQYYAMKILDK-AKEDFLR-a9-b2 | Cβ | Cβ | 72 | 23 | 0 | 28.58 |
| GTYDILVTKDNQTR-IIVKNNAK-a9-b4 | RIIα | RIIα | 186 | 239 | 0.49 | 28 |
| VADAKGDSESEEDEDLEVPVPSR-KMFESFIESVPLLK-a5-b1 | RIIα | RIIα | 71 | 246 | 0 | 27.74 |
| SNKDGGNQEVEIAR-EFLAKAK-a3-b5 | RIIα | Cβ | 314 | 21 | 0 | 27.72 |
| SNKDGGNQEVEIAR-IVSGKVR-a3-b5 | RIIα | Cβ | 314 | 254 | 0 | 27.63 |
| VIHPKTDEQR-IVSGKVR-a5-b5 | RIIα | Cβ | 118 | 254 | 0 | 27.54 |
| QSLGHPPPEPGPDRVADAKGDSESEEDEDLEVPVPSR-NLLQVDLTKR-a19-b9 | RIIα | Cβ | 71 | 279 | 0 | 27.5 |
| VADAKGDSESEEDEDLEVPVPSRFNR-GTYDILVTKDNQTR-a5-b9 | RIIα | RIIα | 71 | 186 | 0.46 | 27.31 |
| IVKADEHVIDQGDDGDNFYVIER-VADAKGDSESEEDEDLEVPVPSR-a3-b5 | RIIα | RIIα | 157 | 71 | 0.71 | 27.25 |
| NISHYEEQLVKMFGSSVDLGNLGQ-KMFESFIESVPLLKSLEVSER-a11-b14 | RIIα | RIIα | 387 | 259 | 0.46 | 27.15 |
| QIEHTLNEKR-IIVKNNAK-a9-b4 | Cβ | RIIα | 92 | 239 | 0 | 26.99 |
| QSLGHPPPEPGPDRVADAKGDSESEEDEDLEVPVPSR-MKIVDVIGEK-a19-b2 | RIIα | RIIα | 71 | 268 | 0 | 26.92 |
| HKATEQYYAMK-ILDKQK-a2-b4 | Cβ | Cβ | 63 | 76 | 0.7 | 26.86 |
| VADAKGDSESEEDEDLEVPVPSR-FPSHFSSDLKDLLR-a5-b10 | RIIα | Cβ | 71 | 266 | 0.88 | 26.68 |
| KMFESFIESVPLLK-IIVKNNAKK-a1-b8 | RIIα | RIIα | 246 | 243 | 0 | 26.53 |
| VRFPSHFSSDLKDLLR-KVEAPFIPK-a12-b1 | Cβ | Cβ | 266 | 309 | 0 | 26.51 |
| AKEDFLR-IVSGKVR-a2-b5 | Cβ | Cβ | 23 | 254 | 0 | 26.47 |
| LKQIEHTLNEKR-NGVSDIKTHK-a11-b7 | Cβ | Cβ | 92 | 292 | 0 | 26.44 |
| LKQIEHTLNEK-AKEDFLR-a2-b2 | Cβ | Cβ | 83 | 23 | 0 | 26.43 |
| VADAKGDSESEEDEDLEVPVPSR-VRFPSHFSSDLKDLLR-a5-b12 | RIIα | Cβ | 71 | 266 | 0 | 26.3 |
| ATEQYYAMKILDK-LKQIEHTLNEKR-a9-b11 | Cβ | Cβ | 72 | 92 | 0.34 | 26.27 |
| LEYSFKDNSNLYMVMEYVPGGEMFSHLR-LKQIEHTLNEKR-a6-b11 | Cβ | Cβ | 111 | 92 | 0 | 26.18 |
| NLLQVDLTKR-AKEDFLR-a9-b2 | Cβ | Cβ | 279 | 23 | 0 | 26.16 |
| NLLQVDLTKRFGNLK-NGVSDIKTHK-a9-b7 | Cβ | Cβ | 279 | 292 | 0 | 26.1 |
| IVKADEHVIDQGDDGDNFYVIER-IIVKNNAK-a3-b4 | RIIα | RIIα | 157 | 239 | 0.2 | 25.73 |
| VADAKGDSESEEDEDLEVPVPSR-MKIVDVIGEK-a5-b2 | RIIα | RIIα | 71 | 268 | 0 | 25.15 |
| VMLVKHK-ILDKQK-a5-b4 | Cβ | Cβ | 61 | 76 | 0 | 25.13 |
| DLKPENLLIDHQGYIQVTDFGFAKR-KVEAPFIPK-a24-b1 | Cβ | Cβ | 189 | 309 | 0.37 | 24.94 |
| IVKADEHVIDQGDDGDNFYVIER-AKEDFLR-a3-b2 | RIIα | Cβ | 157 | 23 | 0 | 24.77 |
| HKATEQYYAMKILDK-QKVVKLK-a11-b2 | Cβ | Cβ | 72 | 78 | 0.95 | 24.56 |
| KMFESFIESVPLLK-RIIVKNNAK-a1-b5 | RIIα | RIIα | 246 | 239 | 0.86 | 24.36 |
| GTYDILVTKDNQTR-LKQIEHTLNEKR-a9-b11 | RIIα | Cβ | 186 | 92 | 0.46 | 23.96 |
| GTYDILVTKDNQTR-LKQIEHTLNEKR-a9-b2 | RIIα | Cβ | 186 | 83 | 0.52 | 23.87 |
| LEYSFKDNSNLYMVMEYVPGGEMFSHLR-LKQIEHTLNEKR-a6-b2 | Cβ | Cβ | 111 | 83 | 0.17 | 23.64 |
| KWENPPPSNAGLEDFER-KVEAPFIPK-a1-b1 | Cβ | Cβ | 29 | 309 | 0 | 23.61 |
| DLKPENLLIDHQGYIQVTDFGFAKR-AKEDFLRK-a24-b2 | Cβ | Cβ | 189 | 23 | 0 | 23.48 |
| RKMFESFIESVPLLK-ILDKQK-a2-b4 | RIIα | Cβ | 246 | 76 | 0 | 23.14 |
| VADAKGDSESEEDEDLEVPVPSRFNR-VRFPSHFSSDLKDLLR-a5-b12 | RIIα | Cβ | 71 | 266 | 0.48 | 23.09 |
| NISHYEEQLVKMFGSSVDLGNLGQ-VADAKGDSESEEDEDLEVPVPSR-a11-b5 | RIIα | RIIα | 387 | 71 | 0 | 22.63 |
| VADAKGDSESEEDEDLEVPVPSR-SNKDGGNQEVEIAR-a5-b3 | RIIα | RIIα | 71 | 314 | 0 | 22.09 |
| ATEQYYAMKILDKQK-LKQIEHTLNEKR-a9-b2 | Cβ | Cβ | 72 | 83 | 0.7 | 21.98 |
| ILDKQK-NNAKKR-a4-b4 | Cβ | RIIα | 76 | 243 | 0.74 | 21.72 |
| VADAKGDSESEEDEDLEVPVPSR-KVEAPFIPKFR-a5-b9 | RIIα | Cβ | 71 | 317 | 0 | 21.64 |
| QSLGHPPPEPGPDRVADAKGDSESEEDEDLEVPVPSR-IVSGKVR-a19-b5 | RIIα | Cβ | 71 | 254 | 0.21 | 21.59 |
| KMFESFIESVPLLKSLEVSER-RIIVKNNAK-a1-b5 | RIIα | RIIα | 246 | 239 | 0.32 | 21.46 |
| LKQIEHTLNEKR-AKEDFLR-a11-b2 | Cβ | Cβ | 92 | 23 | 0 | 21.31 |
| VADAKGDSESEEDEDLEVPVPSR-IVDVIGEKIYKDGER-a5-b8 | RIIα | RIIα | 71 | 276 | 0 | 21.24 |
| DLKPENLLIDHQGYIQVTDFGFAKR-KKTLGTGSFGR-a24-b2 | Cβ | Cβ | 189 | 47 | 0 | 21.22 |
| LKQIEHTLNEK-ILDKQKVVK-a2-b4 | Cβ | Cβ | 83 | 76 | 0.95 | 21.12 |
| KWENPPPSNAGLEDFER-NGVSDIKTHK-a1-b7 | Cβ | Cβ | 29 | 292 | 0.27 | 20.79 |
| IVKADEHVIDQGDDGDNFYVIER-IYKDGER-a3-b3 | RIIα | RIIα | 157 | 279 | 0.38 | 20.34 |
| DLKPENLLIDHQGYIQVTDFGFAKR-KKTLGTGSFGR-a24-b1 | Cβ | Cβ | 189 | 46 | 0.23 | 20.09 |
The first column lists the amino acid sequence of cross-linked peptides and the positions of the cross-linked lysine residues. DeltaS is a measure for how close the best assigned hit was scored in regard to the second best. The peptides are listed in order of linear discriminant score, which is a weighted sum of the difference used to assess the quality of the composite MS2 spectrum as calculated by xQuest.
Quantitative XL-MS Confirms the Position of the RIIβ D/D.
We next quantified how addition of AKAP18α altered the abundance of cross-links within RIIβ–Cβ. We anticipated that dynamic links would help to establish where the AKAP binds and if it alters the conformation of RIIβ–Cβ. We used the xTract algorithm (38) to identify changes in the abundance of 45 unique cross-linked sites that we were able to reliably quantify over the different replicate samples (Table S5). Five cross-linking sites decreased to less than one-half their original abundance on addition of AKAP18α (Fig. 4D, red links and Table 1). The greatest decrease was observed for a link between RIIβ lysines 285 and 333 (5.4-fold decrease, P = 1.32 × 10−9) (Fig. 4D). The four other decreasing links involved conjugation of RIIβ K46 to lysines either within (RIIβ lysines 263, 285, and 357) or adjacent to (Cβ K266) RIIβ CNBB (red in Fig. 4D). This pattern of down-regulated links confirms that the D/D domain of RIIβ is between its CNBB domains: binding of AKAP18α to the D/D domain sterically impedes DSS from bridging between lysines in this region of PKA (Fig. 4D). Surprisingly, three cross-links were increased by more than 50% (P < 0.01) on addition of AKAP18α (Fig. 4E, purple and Table 1). All three links fall within the C-subunit N lobe, suggesting that binding of AKAP18α propagates a structural rearrangement in this region that enables DSS to cross-link some N-lobe lysines more efficiently. Two of the up-regulated links involve lysines within the C-subunit A helix (lysines 16 and 21 in Fig. 4E), which is a locus for posttranslational modification, including myristylation. The N terminus of AKAP18α, which is separated by ∼20 amino acids from the anchoring helix, is also lipidated at three sites for insertion into the cell membrane. RII subunits, anchored to this AKAP at least, are, therefore, likely to orient C subunits with the myristylated A helices pointing toward the cell membrane (Fig. 5).
Table 1.
| Dynamic cross-link | Ratio (±AKAP18α) | P value |
|---|---|---|
| Decreased abundance + AKAP18α | ||
| RIIβ K285–RIIβ K324 | 0.186 | 1.32 × 10−9 |
| Cβ K266–RIIβ K46 | 0.345 | 2.58 × 10−3 |
| RIIβ K263–RIIβ K46 | 0.387 | 1.58 × 10−5 |
| RIIβ K285–RIIβ K46 | 0.424 | 1.3 × 10−6 |
| RIIβ K357–RIIβ K46 | 0.462 | 1.17 × 10−4 |
| Increased abundance + AKAP18α | ||
| Cβ 16–Cβ 292 | 2.12 | 1.26 × 10−6 |
| Cβ 21–Cβ 83 | 1.92 | 2.23 × 10−4 |
| Cβ 76–Cβ 83 | 1.64 | 4.07 × 10−6 |
Unique cross-linking sites are listed if abundance either increased by more than 50% or decreased to less than 50% on addition of AKAP18α. P was < 0.01 for all sites. Data for all 45 unique cross-linking sites that could be reliably quantified are listed in Table S5.
Fig. 5.
Table S5.
| Protein A | Protein B | Position A | Position B | Log2 ratio (+18α/−18α) | P value |
|---|---|---|---|---|---|
| RIIβ | RIIβ | 285 | 324 | −2.428 | 1.32E-09 |
| Cβ | RIIβ | 266 | 46 | −1.536 | 0.0025818 |
| RIIβ | RIIβ | 263 | 46 | −1.37 | 1.58E-05 |
| RIIβ | RIIβ | 285 | 46 | −1.237 | 1.30E-06 |
| RIIβ | RIIβ | 357 | 46 | −1.113 | 0.000116665 |
| Cβ | RIIβ | 192 | 263 | −1.096 | 0.0120618 |
| RIIβ | RIIβ | 174 | 46 | −0.655 | 0.00032057 |
| Cβ | Cβ | 192 | 23 | −0.652 | 1.65E-05 |
| Cβ | RIIβ | 81 | 285 | −0.646 | 0.00159942 |
| RIIβ | RIIβ | 326 | 357 | −0.608 | 0.052444 |
| RIIβ | RIIβ | 256 | 263 | −0.547 | 0.062685 |
| RIIβ | RIIβ | 256 | 285 | −0.433 | 0.00022393 |
| RIIβ | RIIβ | 260 | 263 | −0.426 | 0.042998 |
| RIIβ | RIIβ | 260 | 285 | −0.408 | 0.035763 |
| RIIβ | RIIβ | 135 | 256 | −0.39 | 0.035824 |
| Cβ | Cβ | 254 | 279 | −0.362 | 0.021884 |
| Cβ | Cβ | 23 | 309 | −0.332 | 0.0020576 |
| RIIβ | RIIβ | 324 | 326 | −0.316 | 0.23174 |
| Cβ | RIIβ | 254 | 174 | −0.285 | 0.0066222 |
| Cβ | Cβ | 47 | 61 | −0.201 | 0.35244 |
| RIIβ | RIIβ | 174 | 256 | −0.167 | 0.40362 |
| RIIβ | RIIβ | 324 | 328 | −0.145 | 0.43816 |
| Cβ | Cβ | 23 | 92 | −0.033 | 0.69412 |
| Cβ | Cβ | 76 | 81 | −0.029 | 0.92618 |
| Cβ | Cβ | 292 | 309 | 0.025 | 0.86738 |
| Cβ | Cβ | 317 | 47 | 0.041 | 0.5802 |
| Cβ | Cβ | 292 | 29 | 0.068 | 0.76764 |
| RIIβ | RIIβ | 135 | 285 | 0.076 | 0.67778 |
| Cβ | Cβ | 317 | 46 | 0.105 | 0.173977 |
| Cβ | Cβ | 81 | 92 | 0.176 | 0.130277 |
| Cβ | Cβ | 23 | 292 | 0.229 | 0.15416 |
| Cβ | Cβ | 23 | 61 | 0.251 | 0.37134 |
| Cβ | Cβ | 292 | 92 | 0.262 | 0.37174 |
| Cβ | Cβ | 76 | 92 | 0.328 | 0.00040339 |
| Cβ | Cβ | 78 | 83 | 0.336 | 0.044683 |
| RIIβ | RIIβ | 328 | 357 | 0.336 | 0.000160641 |
| RIIβ | RIIβ | 135 | 174 | 0.36 | 0.00058064 |
| Cβ | Cβ | 72 | 92 | 0.488 | 0.084118 |
| Cβ | Cβ | 29 | 76 | 0.558 | 0.123624 |
| Cβ | Cβ | 61 | 72 | 0.62 | 0.048564 |
| Cβ | Cβ | 72 | 81 | 0.699 | 0.0199013 |
| Cβ | Cβ | 76 | 83 | 0.715 | 4.07E-06 |
| Cβ | Cβ | 23 | 83 | 0.939 | 0.00022252 |
| Cβ | Cβ | 72 | 83 | 0.971 | 0.011418 |
| Cβ | Cβ | 16 | 292 | 1.086 | 1.26E-06 |
The table lists the relative change in cross-linking between different lysine pairs within RIIβ–Cβ on inclusion of AKAP18α. Lysine pairs are listed starting with the link that was most reduced by inclusion of AKAP18α. Changes in abundance are expressed as log2 (abundance with AKAP18α/abundance without AKAP18α). P value indicates the regression between the two conditions.
Discussion
This study presents three sets of experiments that clarify how PKA C subunits are controlled in cells. First, experiments using SDA show that C subunits are released from both RI and RII during cAMP elevation, suggesting that tethering to R subunits during cAMP activation does not constitute a cellular mechanism for restricting C-subunit activity. This is consistent with many in vitro measurements showing cAMP-induced R–C subunit dissociation using methods including scintillation proximity assay and surface plasmon resonance (23, 39) and with FRET changes between microinjected and genetically encoded R and C subunits bearing fluorescent labels (40, 41). Second, subunit quantitation in a range of protein extracts reveals that R subunits typically exist in an ∼17-fold excess of C subunits, with very high concentrations in tissues, including forebrain. High subunit concentrations and ratios heavily skewed toward R subunits will support high rates of R–C association in cells, thereby limiting the distance from point of release over which C subunits can phosphorylate substrates. Typically, membrane-associated RII subunits outnumber RI subunits by ∼2:1. XL-MS analysis of PKA tetramers containing RII subunits (Fig. 5 and Fig. S4) suggests that, in general, anchored type II isozymes orient with the N terminus of the C subunit pointing toward the anchoring site. In the case of AKAP18α–RIIβ–Cβ, this architecture suggests that myristate (yellow in Fig. 5) and palmitate (pink in Fig. 5) groups attached at the N termini of AKAP18α (black in Fig. 5), and the C subunit (green in Fig. 5) could simultaneously insert into the cell membrane in type IIβ holoenzymes anchored to this AKAP.
We found that PKA subunit concentrations and ratios vary with tissue type (Fig. 3B). The four PKA R-subunit isoforms are structurally and functionally different. Studies with genetically modified mice (42) suggest that RIIβ is more functionally critical than RIIα. Type IIβ tetramers are also more compact (1) and less sensitive to cAMP (37) compared with RIIα. We found that RIIβ subunits are the predominant PKA subunit in forebrain, whereas RIIα subunits predominate in lungs and skeletal muscle (Fig. 3B). It should be noted that, within the forebrain, RIIα and RIIβ exhibit marked neuron-specific patterns of expression (43). Genetic studies suggest that RI is more important than RII for regulating nuclear C-subunit entry and concomitant gene expression (1, 23, 43). We found that RI subunits outnumber RII subunits in only heart and cerebellar extracts (Fig. 3B). Cardiac myxoma is a common symptom of Carney complex, which is caused in most cases by inactivation of the gene coding for RIα (5, 44). This is consistent with a prominent role for RI subunits in inhibiting cardiac C subunits. Anchored type II PKA isozymes are thought to be responsible for rapid PKA signaling processes, including ion channel regulation (15). Consistent with this model, we found very high expression levels of RII subunits in forebrain. Our XL-MS measurements show that anchoring of RIIβ and potentially, RIIα subunits is compatible with membrane insertion of C subunits within anchored type II tetramers. Substrate concentration affects the cAMP sensitivity of type I—but not type II—PKA holoenzymes (45). The insensitivity of RII subunits to substrate concentration may support rapid C-subunit binding and release within the plane of neuronal membranes where local substrate concentrations are high. Membrane tethering of C subunits released from RII subunits could potentially explain why nuclear C-subunit activity is more dependent on RI subunits (23, 43). Consistent with this model, binding of C subunits to RII but not RI favors the myr-out conformation that enables efficient C-subunit membrane insertion (12). A-kinase interacting protein 1 (AKIP1) and protein kinase inhibitor peptide (PKI) will also influence the cellular localization of C subunits. AKIP1 is nuclear (46) and binds to the N terminus of the PKA C subunit, whereas the nuclear localization of PKI is cell cycle-dependent (47). PKI could potentially inhibit ≥20% C in some neurons, but there is uncertainty regarding its exact concentration in brain extracts (47); therefore, it is difficult to relate our calculated concentrations for R and C subunits to PKI.
In vitro binding studies have previously shown that the fraction of 30 nM Cα bound to RIα rises with increasing [RIα] in the presence of 50 μM cAMP, with Kd = 0.24 μM (23). Our estimates for PKA subunit concentrations in HEK293T cells (2 μM RII, 0.7 μM RI, and 0.2 μM C) show that PKA subunits are present at substantially higher concentrations than this Kd in cells. Consistent with a prediction by Kopperud et al. (23) that a minority of C subunits remain associated with R subunits during maximal cAMP elevation in cells, we detected residual RI–C/RII–C cross-linking on strong β-AR stimulation at ∼16/22% of the basal cross-linking intensity according to semiquantitative densitometry (Fig. 1E and Fig. S1E). In comparison, addition of exogenous 10 μM cAMP to postlysis material led to a more pronounced reduction in RII–C cross-linking (Fig. S1 C and D). Therefore, the residual R–C cross-linking observed in cells probably represents partial R–C association during strong β-AR stimulation of HEK293T cells. Together, our subunit quantitation and SDA measurements suggest that abundant R subunits support high rates of R–C association, such that even maximal β-AR stimulation does not fully dissociate R and C subunits.
The phosphorylation state of RII subunits is also emerging as an important determinant of the rate at which C subunits bind to RII subunits (14). A recent study quantified kon constants for C-subunit binding to RIIα using surface plasmon resonance (14). Remarkably, dephosphorylation of RIIα Ser112 increased the kon coefficient for C-subunit binding by 60-fold (14). We detected molar excesses of RII subunits relative to C subunits in every rat extract tested (Fig. 3B). Therefore, typically, the autoinhibitory sequences of most RII subunits will be unencumbered by C subunits and accessible to cellular phosphatases. Phosphatase access is not relevant to RI, since the autoinhibition sequence of these regulatory subunits contains an alanine at the equivalent position to Ser112 (1). A recent study showed that U2OS cells expressing a fusion of RIIα and Cα subunits (“R2C2”), in place of endogenous Cα and RII subunits, exhibit PKA activity according to a cytoplasmic AKAR4 reporter after isoproterenol stimulation (21). Nuclear AKAR4 responses are blunted in cells expressing the R2C2 fusion (21). A possible explanation for these findings is that residues corresponding to the C subunit are still able to sufficiently dissociate from the regulatory elements of RII within the context of the fused R2C2 polypeptide to phosphorylate AKAR4 in the cytosol. An analogy would be activation of other AGC protein kinases by dissociation of regulatory and catalytic elements within a single polypeptide, such as in activation of protein kinase G (22). The artificially high (equimolar) ratio of C to RII and potentially raised RII phosphorylation in R2C2 may counterbalance reductions in cAMP-induced dissociation caused by fusing the two subunits within a single polypeptide. Since the C subunit cannot diffuse away from RII after dissociation in the context of R2C2, it will be unable to pass into the nucleus, consistent with blunted nuclear AKAR4 responses in cells expressing R2C2 (21). Nevertheless, these experiments suggest that, at least in the cytosol, PKA phosphorylation can be achieved with diffusion of the C subunit over a very short distance from RII.
Rapid fluctuations in local cAMP concentration, supported by colocalization of PKA with adenylyl cyclases and phosphodiesterases, will also support faster release and binding of C subunits to R subunits. For example, type IV phosphodiesterases (PDEs) can anchor to AKAPs (48), and direct PKA–PDE coupling has been detected (49). XL-MS is developing rapidly, and it is plausible that the technique could be applied to study cAMP signaling complexes in cells. In the future, it will be exciting to determine how anchored PKA complexes are oriented in relation to interaction partners, including cyclases, phosphodiesterases, and receptors (50).
Materials and Methods
For quantitative immunoblotting, samples were collected from HEK293Ts and male 4-wk-old Sprague–Dawley rats and homogenized using a DI 25 Basic rotor/stator homogenizer (Yellowline) and 20-kHz sonication. Experiments involving rats were done in accordance with the United Kingdom Animals Act, 1986 and with University College London Animal Research guidelines. For XL-MS measurements, digested PKA complexes were analyzed on Orbitrap Elite and Fusion Tribrid mass spectrometers (Thermo). For quantitative XL-MS analysis, we compared the intensities of peaks eluting for cross-links between the RIIβ–Cβ and RIIβ–Cβ–AKAP18α samples using xTract (38). Results are presented throughout as mean ± SEM. Data were analyzed by two-sided Student’s t test. P values are *P < 0.05, **P < 0.01, and ***P < 0.001. Detailed methods can be found in SI Materials and Methods.
SI Materials and Methods
Light-Activated Cross-Linking in HEK293T Cells.
HEK293T cells were cultured as described below. At 80% confluence, cells were resuspended in DMEM from plates totaling 350 cm2 and pelleted by centrifugation at 1,200 × g for 4 min at room temperature. The cells were washed by resuspending in 10 mL PBS and pelleted again by centrifugation for 4 min at 1,200 × g; 10 mM SDA (Thermo Fisher Scientific) was freshly prepared at 10 mM in DMSO, and 1 mL of SDA stock was added to 9 mL PBS for a final concentration of 1 mM. This solution was used to resuspend the pelleted HEK293T cells. The cell suspension was incubated at 22 °C with 1 mM SDA on a roller mixer for 30 min, after which 1 M Tris, pH 7.4, was added to a final concentration of 10 mM to quench unreacted NHS moieties. The cells were pelleted as before, the supernatant was aspirated, and the cells were washed with 10 mL PBS. The cells were next resuspended in 10 mL PBS supplemented with 1 μM isoproterenol if appropriate. Isoproterenol hydrochloride was prepared immediately before experiments by dissolving in PBS to produce 1 mM stock solutions. After 5 min of incubation at 22 °C, cells were next transferred to a 60-cm2 tissue culture dish and illuminated on ice with UV light from a high-intensity Blak-Ray B-100AP 365-nm UV lamp (UVP) positioned ∼10 cm above the surface of the plate. After illumination for 5 min, cells were pelleted as before, the supernatant was aspirated, and the cells were washed in PBS and pelleted once more. The cells were resuspended in 1 mL extraction buffer, sonicated, and centrifuged at 21,100 × g for 1 h at 4 °C. The supernatant was collected for R subunit-selective pull down. For “postlysis” UV-induced cross-linking, UV illumination was delayed until after cell lysis. In these cases, the supernatant was collected and incubated overnight at 4 °C on a rotating mixer. On the following morning, the sample was diluted into 9 mL detergent-free extraction buffer and then subjected to 5 min of UV illumination as above. Postlysis UV illumination samples were supplemented with 10 μM cAMP 10 min before UV illumination as appropriate.
R Subunit-Selective Pull Down.
On mornings in advance of HEK293T SDA cross-linking later in the day, 30-μL aliquots of glutathione Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) were charged with 30 µg GST, GST-RIAD, or GST-AKAP79c93. In each case, bead charging was performed by incubating on a rotating mixer at 4 °C for 2 h in 500 μL pull-down buffer (20 mM Hepes, pH 7.5, 200 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.05% TWEEN 20). The beads were washed with 4× 1 mL pull-down buffer and then transferred to 15-mL falcon tubes in a final volume of 4.5 mL of the same buffer; 1 mL HEK293T cell extracts were divided into two, and 0.5 mL lysate was added to the appropriate charged beads (typically one-half to GST-RIAD and one-half to GST-AKAP79c93 beads). The mixtures were incubated overnight on a rolling mixer at 4 °C. On the following morning, beads were pelleted at 1,200 × g for 3 min, and the supernatant was aspirated. The beads were washed with 5× 1 mL pull-down buffer supplemented with 1 mM cAMP to remove C subunits noncovalently bound to R subunits. After the final wash step, the beads were resuspended in 1× NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT and incubated at 85 °C for 10 min to release GST fusion and associated proteins. Covalent attachment of C subunits to RI or RII was assessed by immunoblotting using anti-Pan C antibody. Densitometry was performed in the linear range of the antibody when performing quantitative comparisons.
To test if GST-RIAD is capable of pulling down both RIα and RIβ, 20-μL aliquots of glutathione Sepharose beads, charged with 12 μg GST-RIAD, were incubated with 1 mL extraction buffer containing 0.11 ng/μL either RIα or RIβ. After overnight incubation at 4 °C on a spinning mixer, the beads were washed with 5× 200 μL extraction buffer, and protein was released from the glutathione Sepharose as above. Pull-down efficiency was assessed by anti-Pan RI antibody IB.
Protein Expression and Purification.
All PKA subunits purified during this investigation correspond to the human sequences. RII lysines are numbered according to the equivalent positions in crystallized mouse sequences to facilitate cross-referencing with crystal structures (2QVS for RIIα, 3TNP for RIIβ). PKA Cβ, RIα, RIβ, RIIα, RIIβ, AKAP18α, AKAP79 (335–427), and RIAD were expressed as PreScission-cleavable fusions at the C terminus of GST using the vector pGEX6P1. PKA Cα was expressed with an N-terminal 6xHis tag using pET28a. All proteins were expressed in Escherichia coli BL21 (DE3) cells by inducing cultures of 1.6–4.8 L at an OD600 ∼ 0.7 with 375 μM isopropyl β-d-1-thiogalactopyranoside. After incubation with shaking at 20 °C overnight, cells were pelleted by centrifugation for 10 min at 4,000 × g. Cell pellets were washed in PBS and frozen at −80 °C before processing. All protein purification steps were performed on ice. For cell lysis, cell pellets were thawed in 50–100 mL of either glutathione Sepharose binding buffer (30 mM Hepes, pH 7.5, 500 mM NaCl, 2 mM DTT, 0.5 mM EDTA, 1 mM benzamidine) or Ni-NTA binding buffer (30 mM Tris, pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM benzamidine) as appropriate. All lysates were supplemented with 0.1 mg/mL lysozyme; lysates for purifying AKAP18α, Cβ, RIIβ, and RIα were supplemented with 10% glycerol; the AKAP18α lysate was supplemented with 0.5% Triton TX-100; and the Cα lysate was supplemented with one Complete EDTA-free protease inhibitor tablet (Roche) per 100 mL lysate. Lysates were sonicated at 20 kHz for 2 × 30 s and then mixed on a roller for 30 min before centrifugation at 31,360 × g for 30 min. Clarified supernatants were incubated for either 3 h with 2 mL glutathione Sepharose 4B (GE Healthcare Life Sciences) or 1 h with 2 mL Ni-NTA agarose (Qiagen) as appropriate. After binding, affinity beads were washed with 3× 10 mL binding buffer before elution.
Elution from glutathione Sepharose was performed in two ways depending on whether cleavage from GST was desired. For GST fusion proteins, elution was performed by incubating for 1 h with 2 mL of binding buffer supplemented with 10 mM l-glutathione. For release of purified PKA subunit alone, beads were incubated with 60 μg PreScission protease (GE Healthcare Life Sciences) in 2 mL glutathione Sepharose binding buffer. In this case, elution was performed by overnight incubation at 4 °C. Elution of His-tagged Cα subunits from Ni-NTA agarose was performed in batch by incubating with Ni-NTA elution buffer (30 mM Tris, pH 7.0, 500 mM NaCl, 300 mM imidazole, 1 mM benzamidine). In the final purification step, all protein samples were separated by size exclusion chromatography (SEC) using an 80-mL Superdex 200 column (GE Healthcare Life Sciences) controlled by an ÄKTA fast protein liquid chromatography system (GE Healthcare Life Sciences). SEC was performed with Hepes-NaCl buffer (20 mM Hepes, pH 7.5, 200 mM NaCl). High-purity protein was collected from peak fractions on the basis of Coomassie staining fractions separated by gel electrophoresis on 4–12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific).
Preparation of Protein Extracts.
Male 4-wk-old Sprague–Dawley rats were euthanized by cervical dislocation, and organs were removed immediately. Forebrain, cerebellum, heart, tibialis anterior muscle, lung, and liver were excised on ice, washed in PBS, and weighed. Extraction buffer [30 mM Hepes, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM benzamidine, 1% (wt/vol) Igepal CA-630, 0.25% (wt/vol) sodium deoxycholate] was added to each sample at 1 mL per 100 mg wet mass. The organs were manually sliced into smaller pieces before homogenization at 9,500 rpm using a Di 25 basic rotor/stator homogenizer (Yellowline) for 2 × 30 s. The homogenate was next sonicated for 30 s at 20 kHz and finally clarified by centrifugation at 21,130 × g for 1 h at 4 °C. The resulting supernatant was collected for quantitative immunoblotting and measurement of total protein content using bicinchoninic acid (BCA) assay. The concentration of protein in extracts was determined by BCA assay (milligrams per milliliter): cerebellum (6.68 ± 0.70), forebrain (8.52 ± 0.25), heart (7.73 ± 0.71), liver (13.50 ± 1.38), lung (8.21 ± 1.15), skeletal muscle (5.71 ± 0.48), and nerve ending-rich forebrain fraction (8.83 ± 0.48). Nerve ending-enriched fractions were collected using Dounce homogenization to fraction S1. Briefly, rat forebrains were excised, rinsed in ice cold sucrose/EDTA buffer (0.32 M sucrose, 1 mM EDTA, 5 mM Hepes, pH 7.4), weighed, and resuspended in 3.5 mL sucrose/EDTA buffer. After coarse manual chopping, the sample was processed using a Dounce homogenizer (five strokes with loose insert followed by five strokes with tight insert). The homogenate was centrifuged at 1,000 × g for 10 min, and the resulting supernatant was collected for analysis. BCA assay indicated that this protocol yields ∼30 mg protein per 1 g starting wet mass.
In advance of preparation of protein extracts, HEK293T cells were cultured in Dulbecco's modified eagle media (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 10% FBS, and 2 mM glutaMAX at 37 °C in 5% CO2. All tissue culture media and additives were supplied by Gibco. Cells at ∼70% confluence on T75 plates were washed and resuspended in PBS, and cell number per milliliter was determined using a hemocytometer to enable the average number of cells per microgram total protein extract to be calculated later in the analysis. Next, the cells were pelleted at 1,200 × g, resuspended in 700 μL extraction buffer, and sonicated for 30 s at 20 MHz. The lysate was clarified by centrifugation for 1 h at 21,130 × g. The supernatant was collected for immunoblotting and analysis/BCA assay.
Quantitative Immunoblotting.
Immunoblotting was performed using mouse anti-PKA subunit primary antibodies purchased from BD Biosciences. Antibodies were diluted to 0.8 μg/mL in Tris-buffered saline + 0.05% Tween-20 supplemented with 10% (wt/vol) milk, with the exception of Pan C antibody (0.5 μg/mL). Experiments were performed using antibodies recognizing C subunits (detection range 0.1–100 ng; product no. 610981), RI subunits (1–100 ng; product no. 610166), RIα (product no. 610609), RIIα (3–50 ng; product no. 612243), and RIIβ (3–50 ng; product no. 610626). Primary antibodies were coupled to goat anti-mouse IgG (H+L) poly-HRP secondary antibody (Thermo Fisher Scientific), and peroxidase was detected with SuperSignal West Dura substrate (Thermo Fisher Scientific) using an ImageQuant imaging unit (GE Healthcare Life Sciences). Band intensities for reference protein standards, and experimental samples were calculated using ImageJ (NIH) for background subtraction and pixel integration. For each IB, reference curves were established by fitting to Hill functions using iterative least squares refinement with the Levenberg–Marquardt algorithm in ORIGIN software (OriginLab). Fitted curves (with typical R2 coefficients > 0.99) were used to determine the protein concentration of the given PKA subunit in experimental samples transferred on the same nitrocellulose membrane. Calibration experiments were performed to determine optimal sample loading volumes and exposure times that generated signal within the dynamic range of the relevant antibody before the full analysis (n = 4 for each subunit in each extract) was completed.
XL-MS.
RIIα–Cβ, RIIβ–Cβ, and RIIβ–Cβ–AKAP18α complexes were cross-linked in 100 μL Hepes-NaCl buffer composed of 50 μg RIIα + 50 μg Cβ, 50 μg RIIβ + 50 μg Cβ, and 50 μg RIIβ + 50 μg Cβ + 5 μg AKAP18α, respectively. The samples were preincubated at 30 °C for 10 min shaking at 500 rpm in a Thermomixer (Eppendorf). Cross-linking was initiated by addition of 0.5 mM H12/D12 DSS (Creative Molecules) from a 25 mM stock prepared in DMSO. After another 30 min of shaking at 30 °C, cross-linking was quenched by addition of 50 mM ammonium bicarbonate from a 1 M stock. During analysis of electrophoretically separated cross-linked samples, AKAP18α was detected by far-Western blotting with V5-tagged RIIα subunits coupled to anti-V5-HRP antibody (Thermo Fisher Scientific). Before analysis on the mass spectrometer, cross-linked samples were treated with a urea/Tris(2-carboxyethyl)phosphine solution to reduce disulphide bonds, after which free cysteines were alkylated with iodoacetimide. Protein was next digested with trypsin, and the resulting trypsinized peptide mixture was treated with formic acid to inhibit trypsin activity. Digested peptides were separated from the solution, retained by a solid-phase extraction system, and then separated by SEC before liquid chromatography–MS/MS analysis on Thermo Scientific Orbitrap Elite (RIIβ-containing samples) or Orbitrap Fusion Tribrid (RIIα-containing samples) mass spectrometers. Data were searched using xQuest in ion-tag mode with a precursor mass tolerance of 10 ppm. For matching of fragment ions, tolerances of 0.2 Da for common ions and 0.3 Da for cross-link ions were applied. Cross-linked samples were prepared in duplicate for all complexes, and each of these was measured with technical duplicates. Cross-links were only considered during structural analysis if they registered linear discriminant scores >20 and deltaS < 0.95.
Quantitative XL-MS.
For quantitative XL-MS analysis, we compared the intensities of peaks eluting for cross-links between the RIIβ–Cβ and RIIβ–Cβ–AKAP18α samples (n = 2; each sample analyzed in duplicate). Amounts of potential cross-links were normalized before MS by measuring peptide bond absorption at 215 nm for each fraction. Only high-confidence cross-links that were identified in all biological experiments were selected for additional quantitative analysis. Quantities of identified high-quality cross-links were determined by xTract (37).
Statistical Analysis.
Results are presented throughout as mean ± SEM. Data were analyzed by two-sided Student’s t test. P values are *P < 0.05, **P < 0.01, and ***P < 0.001.
Acknowledgments
We thank Annette Dolphin for use of her cell culture facility and Kanchan Chargar for assistance with cell culture. F.S. is funded by German Research Foundation (DFG) Emmy Noether Programme STE 2517/1-1 and grateful for support from the DFG Collaborative Research Center 969. M.G.G. is Wellcome Trust and Royal Society Sir Henry Dale Fellow 104194/Z/14/Z and receives support from Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N015274/1.
Supporting Information
Supporting Information (PDF)
- Download
- 1.08 MB
References
1
SS Taylor, R Ilouz, P Zhang, AP Kornev, Assembly of allosteric macromolecular switches: Lessons from PKA. Nat Rev Mol Cell Biol 13, 646–658 (2012).
2
K McCormick, GS Baillie, Compartmentalisation of second messenger signalling pathways. Curr Opin Genet Dev 27, 20–25 (2014).
3
JA Esteban, et al., PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat Neurosci 6, 136–143 (2003).
4
JD Corbin, PH Sugden, TM Lincoln, SL Keely, Compartmentalization of adenosine 3′:5′-monophosphate and adenosine 3′:5′-monophosphate-dependent protein kinase in heart tissue. J Biol Chem 252, 3854–3861 (1977).
5
LS Kirschner, et al., Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 26, 89–92 (2000).
6
F Beuschlein, et al., Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome. N Engl J Med 370, 1019–1028 (2014).
7
MG Gold, T Gonen, JD Scott, Local cAMP signaling in disease at a glance. J Cell Sci 126, 4537–4543 (2013).
8
N Patel, MG Gold, The genetically encoded tool set for investigating cAMP: More than the sum of its parts. Front Pharmacol 6, 164 (2015).
9
GC Mo, et al., Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat Methods 14, 427–434 (2017).
10
EC Gaffarogullari, et al., A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes. J Mol Biol 411, 823–836 (2011).
11
SE Tillo, et al., Liberated PKA catalytic subunits associate with the membrane via myristoylation to preferentially phosphorylate membrane substrates. Cell Rep 19, 617–629 (2017).
12
M Gangal, et al., Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch. Proc Natl Acad Sci USA 96, 12394–12399 (1999).
13
P Zhang, et al., Single turnover autophosphorylation cycle of the PKA RIIβ holoenzyme. PLoS Biol 13, e1002192 (2015).
14
P Zhang, et al., An isoform-specific myristylation switch targets type II PKA holoenzymes to membranes. Structure 23, 1563–1572 (2015).
15
P Skroblin, S Grossmann, G Schäfer, W Rosenthal, E Klussmann, Mechanisms of protein kinase A anchoring. Int Rev Cell Mol Biol 283, 235–330 (2010).
16
MG Gold, et al., Molecular basis of AKAP specificity for PKA regulatory subunits. Mol Cell 24, 383–395 (2006).
17
FS Kinderman, et al., A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase. Mol Cell 24, 397–408 (2006).
18
F Götz, et al., AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA. Biochem J 473, 1881–1894 (2016).
19
R Ilouz, et al., Localization and quaternary structure of the PKA RIβ holoenzyme. Proc Natl Acad Sci USA 109, 12443–12448 (2012).
20
FD Smith, et al., Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation. Elife 2, e01319 (2013).
21
FD Smith, et al., Local protein kinase A action proceeds through intact holoenzymes. Science 356, 1288–1293 (2017).
22
LR Pearce, D Komander, DR Alessi, The nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol 11, 9–22 (2010).
23
R Kopperud, et al., Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions. J Biol Chem 277, 13443–13448 (2002).
24
D Willoughby, DMF Cooper, Organization and Ca2+ regulation of adenylyl cyclases in cAMP microdomains. Physiol Rev 87, 965–1010 (2007).
25
CR Carlson, et al., Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor. J Biol Chem 281, 21535–21545 (2006).
26
CJ Mageean, JR Griffiths, DL Smith, MJ Clague, IA Prior, Absolute quantification of endogenous Ras isoform abundance. PLoS One 10, e0142674 (2015).
27
CM Gillen, 3rd B Forbush, Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells. Am J Physiol 276, C328–C336 (1999).
28
HC Chien, et al., Rapid method to determine intracellular drug concentrations in cellular uptake assays: Application to metformin in organic cation transporter 1-transfected human embryonic kidney 293 cells. Drug Metab Dispos 44, 356–364 (2016).
29
NE Erondu, MB Kennedy, Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci 5, 3270–3277 (1985).
30
T Kuno, et al., Distinct cellular expression of calcineurin A alpha and A beta in rat brain. J Neurochem 58, 1643–1651 (1992).
31
YY Huang, XC Li, ER Kandel, cAMP contributes to mossy fiber LTP by initiating both a covalently mediated early phase and macromolecular synthesis-dependent late phase. Cell 79, 69–79 (1994).
32
BW Jones, et al., Targeted deletion of AKAP7 in dentate granule cells impairs spatial discrimination. Elife 5, e20695 (2016).
33
G Cadd, GS McKnight, Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain. Neuron 3, 71–79 (1989).
34
A Leitner, M Faini, F Stengel, R Aebersold, Crosslinking and mass spectrometry: An integrated technology to understand the structure and function of molecular machines. Trends Biochem Sci 41, 20–32 (2016).
35
VC Tibbs, PC Gray, WA Catterall, BJ Murphy, AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels. J Biol Chem 273, 25783–25788 (1998).
36
ID Fraser, et al., A novel lipid-anchored A-kinase anchoring protein facilitates cAMP-responsive membrane events. EMBO J 17, 2261–2272 (1998).
37
P Zhang, et al., Structure and allostery of the PKA RIIβ tetrameric holoenzyme. Science 335, 712–716 (2012).
38
T Walzthoeni, et al., xTract: Software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry. Nat Methods 12, 1185–1190 (2015).
39
FW Herberg, SS Taylor, WR Dostmann, Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase. Biochemistry 35, 2934–2942 (1996).
40
SR Adams, AT Harootunian, YJ Buechler, SS Taylor, RY Tsien, Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697 (1991).
41
M Zaccolo, et al., A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2, 25–29 (2000).
42
LS Kirschner, Z Yin, GN Jones, E Mahoney, Mouse models of altered protein kinase A signaling. Endocr Relat Cancer 16, 773–793 (2009).
43
R Ilouz, et al., Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain. Elife 6, e17681 (2017).
44
CA Stratakis, Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit (PRKAR1A) in patients with the “complex of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas” (Carney complex). Ann N Y Acad Sci 968, 3–21 (2002).
45
K Viste, RK Kopperud, AE Christensen, SO Døskeland, Substrate enhances the sensitivity of type I protein kinase a to cAMP. J Biol Chem 280, 13279–13284 (2005).
46
M Sastri, DM Barraclough, PT Carmichael, SS Taylor, A-kinase-interacting protein localizes protein kinase A in the nucleus. Proc Natl Acad Sci USA 102, 349–354 (2005).
47
GD Dalton, WL Dewey, Protein kinase inhibitor peptide (PKI): A family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function. Neuropeptides 40, 23–34 (2006).
48
KL Dodge, et al., mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 20, 1921–1930 (2001).
49
S Krishnamurthy, et al., Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling. Biophys J 107, 1426–1440 (2014).
50
ML Halls, DM Cooper, Adenylyl cyclase signalling complexes–Pharmacological challenges and opportunities. Pharmacol Ther 172, 171–180 (2017).
51
J Wu, SH Brown, S von Daake, SS Taylor, PKA type IIalpha holoenzyme reveals a combinatorial strategy for isoform diversity. Science 318, 274–279 (2007).
Information & Authors
Information
Published in
Classifications
Submission history
Published online: September 11, 2017
Published in issue: September 26, 2017
Keywords
Acknowledgments
We thank Annette Dolphin for use of her cell culture facility and Kanchan Chargar for assistance with cell culture. F.S. is funded by German Research Foundation (DFG) Emmy Noether Programme STE 2517/1-1 and grateful for support from the DFG Collaborative Research Center 969. M.G.G. is Wellcome Trust and Royal Society Sir Henry Dale Fellow 104194/Z/14/Z and receives support from Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N015274/1.
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Altmetrics
Citations
Cite this article
Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches, Proc. Natl. Acad. Sci. U.S.A.
114 (39) 10414-10419,
https://doi.org/10.1073/pnas.1701782114
(2017).
Copied!
Copying failed.
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.