FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells

FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells

Divisions of ^*Tumor Immunology and ^§Cancer Biology, Dana Farber Cancer Institute, and Departments of ^†Medicine and ^¶Pathology, Harvard Medical School, Boston MA 2115

Communicated by Stuart F. Schlossman, Dana–Farber Cancer Institute, Boston, MA (received for review March 13, 1998)

Abstract

TcRζ/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56^lck, p59^fyn, and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.

Footnotes

↵ ‡ J.L. and H.K. contributed equally to this work.
↵ ‖ To whom reprint requests should be addressed. e-mail: christopher_rudd{at}dfci.harvard.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF072166).
ABBREVIATIONS:

TcR,

T cell receptor;

IL-2,

interleukin 2;

FYB,

FYN binding protein;

SKAP55,

src kinase-associated protein of 55 kDa;

SKAP55R,

SKAP55-related protein;

GST,

glutathione S-transferase;

HA,

hemagglutinin;

FITC,

fluorescein isothiocyanate