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Emaduddin et al. 10.1073/pnas.0712176105.

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Fig. 6. Normalization of protein extracts and detection of basal SFK activity in CRC lines.

(A-D) Analysis of protein amounts in total cell lysates of 64 CRC cell lines by Coomassie blue staining after SDS/PAGE (8.5% gels). The aim is to ensure similar loading in subsequent Western blot analyses. Arrows indicate some high-abundance proteins that vary considerably in their expression between different CRC lines. (E) Western blots of total cell lysate protein extracts from 64 human CRC lines grown as described in Methods. Extracts were separated by SDS/PAGE and immunoblotted with the anti-phosphoTyr419Src antibody that recognizes the SFK members expressed in the CRC lines. Bound anti-phosphoTyr419Src antibody is detected by ECL. The exposure time of the blots to x-ray films was extended to 10 min to also detect weak signals. Note that at least some faint pY419 SFK bands are evident in virtually all CRC lines. Blots with anti-actin as loading control also are shown. It should be noted, however, that total actin levels appear to vary between some lines, although sample loading is very similar according to the Coomassie blue stain of the same protein extracts, presumably reflecting the great degree of protein expression deregulation in some cancer cells, even in the expression of basic structural proteins.

Fig. 7. Sensitivity of CRC cell lines to the SFK inhibitor SU6656. CRC cell lines were treated daily with the indicated concentrations of SU6656 and analyzed as described in Methods. It is noteworthy that SU6656 does not inhibit all SFK members effectively. Published IC₅₀ values for SFK inhibition in vitro are as follows: Yes, 0.02 mM; Lyn, 0.13 mM; Fyn, 0.17 mM; Src, 0.28 mM; Lck, 6.88 mM (1). These in vitro IC₅₀ values correspond typically to »10- to 100-fold higher IC₅₀ values for treatments of cultured cells. CRC aberrantly expressing Lck may therefore be partially resistant to this inhibitor. SU6656 also appears to be less stable than PP2, leading to a reappearance of pTyr proteins after ca. 12 h at least in some lines tested (data not shown).

Fig. 8. Elevated global phosphotyrosine levels in CRC cell lines with high-SFK activity. CRC cell lines were treated and analyzed as described in Methods. Different from a similar experiment in Fig. 3 using the anti-pTyr mAb 4G10, a larger subset of CRC cell lines was analyzed here, and the mAb P-Tyr-100 was used for the detection of pTyr proteins. Note that both of these widely used anti-pTyr mAb result in somewhat different patterns of phosphoproteins being detected, highlighting the well known fact that their target recognition specificity is not identical, and amino acids near the phosphotyrosyl residue also affect binding of the mAb. Interestingly, with the P-Tyr-100 mAb, elevated pTyr levels also are detected in LoVo cells (boxed), thus deviating from the general trend. The actual reason for this deviation is unclear, although LoVo cells have an apparent processing defect for some receptor proteins, for example, for c-Met (Fig. 5) (2) and IGF1R (SI Fig. 10) (3), which may lead to an aberrant accumulation of partially processed receptor tyrosine kinases and subsequently hyperphosphorylation of some cellular proteins independently of SFK activity. The asterisks indicate a nonspecific band very similar in size to actin, which is recognized by P-Tyr-100 in lysates from various cell types. Preincubation of the mAb with soluble pTyr or phenylphosphate eliminates specific phosphoprotein bands, but not this band.

Fig. 9. Reduction of global tyrosine phosphorylation by SFK inhibition in COLO 320DM and DLD-1 cells with high-SFK activity by using SU6656. CRC cell lines were treated for 6 h with DMSO (D) or SU6656 (S) and analyzed as described in Methods. SU6656 is effective against several SFK members (1), but, for example is a very poor inhibitor of Lck (also see SI Fig. 7 legend for further details) and therefore may not show bioactivity in CRC cell lines with substantial aberrant expression of Lck.

Fig. 10. Low steady-state phosphorylation of IGF1R and EGFR in CCC independently of SFK activity status. CRC cell lines indicated were treated and analyzed as described in Methods. Total cell lysates (TCL) or immunoprecipitations (IPs) with indicated antibodies were analyzed by immunoblot (IB). IGF1R and EGFR expression levels vary greatly between cell lines. Arrows indicate the migration of the receptor proteins. (A) No phosphorylation of the key regulatory epitope in IGF1R YY1135/1136 was detected in CCC lines with high-SFK activity. The faint band seen throughout the panel appeared only after prolonged exposure of the blot and was considered to be nonspecific. The asterisk indicates an incompletely processed IGF1R in LoVo cells, which has been reported previously (3). (B) MCF-7 breast cancer cells stimulated with IGF-1 served as a positive control for IGF1R pYpY1135/1136 (data not shown). No steady-state phosphorylation of the EGFR is detectable in high-SFK activity CRC lines. In LoVo cells, several pTyr protein bands are detected in the EGFR-IP. Whether the appearance of these bands is somehow linked to the processing defects and aberrant activation of other receptors like c-Met and IGF1R remains to be determined. The origin of the faint pTyr signals in SW620 cells, which apparently lack EGFR expression according to blots with total cell lysate, also remains elusive. Lysate of EGF-stimulated A431 cells served as a positive control for tyrosine-phosphorylated EGFR (data not shown).

Fig. 11. Inhibition of c-Met kinase activity with SU11274 does not reduce global phosphotyrosine levels or SFK activity. CRC cell lines indicated were treated with 1 mM Met inhibitor SU11274 (M) or DMSO (0.06%; D) for 24 h, and total cell lysates were analyzed with anti-pTyr mAb (P-Tyr-100), anti-pY419Src, and anti-actin. No changes in the overall pTyr levels or SFK activity were observed. IP of c-Met and blotting with pYpY1234/1235-c-Met antibody confirmed the activity of the compound against activated c-Met (data not shown).

Fig. 12. Src activity does not correlate well with the overall activity of SFKs in CRC cells with high-SFK activity. (Middle) Src was immunoprecipitated from total cell lysates of a subset of CRC lines by using the anti-Src mAb 327, which has a strong preference for Src over other SFK members (4) and then analyzed by Western blotting with anti-pTyr419Src for activity. (Top) For ease of comparison, the corresponding signals obtained with total cell lysates for all expressed SFK members by blotting with anti-pTyr419Src (see also Fig. 1) also are shown. (Bottom) The precipitated amount of Src protein also was determined and is shown. The signals obtained for all SFK members are not similar to the Src activity detected in high-SFK lines. Therefore, we conclude that analyzing c-Src or probably any other single SFK family member in isolation will give an incomplete picture of the SFK-mediated signaling events in CRC cells.

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Table 1. CRC cell line panel

*Cell line designations according to cited reference or use by commercial source.

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