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

Abstract

TcRzeta/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.

Figure 1

SKAP55 and SKAP55R clones identified by the two-hybrid screen using FYB as bait. (A) Depiction of full-length FYB and truncated FYB (FYBC) used in the two-hybrid screen. (B) (Left) Expression of FYB and FYBC in yeast. (Right) β-Galactosidase test using full-length FYB identified positive clones Y6-4. Y9-3, Y9-6, Y9-7, Y12-3, Y16-3, Y18-2, and Y48-1. Clones Y13-1 and Y21-3 served as negative controls. (C) Deduced amino acid sequence of the full-length SKAP55R protein. The amino acid sequence of the newly cloned N terminus and midregion insertion is shown in boldface. The PH domain is underlined in boldface, the putative src-SH2 binding motif is underlined, the coiled-coil region is designated by a combination of rectangular box and underline, and the SH3 domain is designated in a rectangular box. (D) Amino acid comparison of SKAP55 and full-length SKAP55R. The amino acid sequence of the newly cloned N terminus and midregion insertion is shown in boldface (upper line). The PH domain of both proteins is underlined in boldface and the SH3 domains are designated in a rectangular box.

Figure 2

SKAP55 and SKAP55R bind to FYB in vivo. (Top) SKAP55 and SKAP55R bind to FYB in COS cells. COS cells were transfected with combinations of FYB, SKAP55, and SKAP55R and assessed for complex formation. Glutathione beads were used to precipitate the GST-fusion proteins, and anti-HA and anti-GST blotting followed. Lane 1, pEBG; lane 2, pEBG and SKAP55R-HA; lane 3, pEBG and SKAP55-HA; lane 4, FYB-GST; lane 5, SKAP55R-HA; lane 6, SKAP55-HA; lane 7, SKAP55R-HA and FYB-GST; and lane 8, SKAP55-HA and FYB-GST. (Middle) Levels of FYB protein expression. As in Top except GST precipitates were blotted with anti-GST. (Bottom) Levels of SKAP55 and SKAP55R expression. Cell lysates were blotted with anti-HA.

Figure 3

SKAP55 and SKAP55R SH3 domains bind to FYB. (A) SKAP55 and SKAP55R SH3 domains bind to FYB in COS cells. COS cells were transfected with combinations of FYB, SKAP55, SKAP55R, and GRB-2 SH3 domains and assessed for complex formation. SH3 domain-containing proteins were expressed as GST fusion proteins. Glutathione beads were used to precipitate the GST-fusion proteins and anti-HA blotting followed. Lane 1, pEBG; lane 2, pEBG and FYB-HA; lane 3, Grb-2 N-terminal SH3 domain; lane 4, Grb-2 C-terminal SH3 domain; lane 5, SKAP55R SH3 domain; lane 6, SKAP55 SH3 domain; lane 7, Grb-2 N-terminal SH3 domain and FYB-HA; lane 8, Grb-2 C-terminal SH3 domain and FYB-HA; lane 9, SKAP55R SH3 domain and FYB-HA; lane 10, SKAP55 SH3 domain and FYB-HA. (B) (Top) SKAP55 and SKAP55R SH3 domains bind to FYB in T cells. DC27.10 cells were transfected with FYB and SKAP55, SKAP55R, and GRB-2 SH3 domains and assessed for complex formation. Lanes as in A. (Middle) Levels of FYB protein expression. As in Top except cell lysate was blotted with anti-HA. (Bottom) Levels of GST-fusion protein expression. As in Top except anti-GST blot.

Figure 4

SKAP55 and SKAP55R act as targets of FYN kinase. (Upper) SKAP55 and SKAP55R act as substrates for FYN. DC27.10 cells were transfected with SKAP55 or SKAP55R and FYN or ZAP-70 and assessed for tyrosine phosphorylation. SKAP55 and SKAP55R were tagged with both GST and HA. GST precipitates were subjected to anti-phosphotyrosine blotting (lanes 7–10). Lane 1, pEBG; lane 2, pEBG and FYN; lane 3, pEBG and ZAP-70; lane 4, SKAP55R-GST-HA; lane 5, SKAP55-GST-HA; lane 6, FYN; lane 7, ZAP-70; lane 8, SKAP55R-GST-HA plus FYN; lane 9, SKAP55-GST-HA plus FYN; lane 10, SKAP55-GST-HA plus ZAP-70. (Lower) Expression levels of SKAP55-GST-HA and SKAP55R-GST-HA. As in Upper except cell lysates were blotted with anti-HA.

Figure 5

Co-localization of FYB with SKAP55 and SLP76 with SKAP55 as detected by confocal immunofluorescence. COS cells transfected with FYB-120, SLP-76, and/or SKAP55 were stained with a combination of FITC- and Texas red-labeled secondary antibodies. (A) FYB and SKAP55 show intracellular colocalization. (Upper Right) Anti-FYB-120. (Lower Right) Anti-SKAP55. (Left) Anti-FYB and anti-SKAP55. (B) SLP-76 and SKAP55 show intracellular colocalization. (Upper Right) Anti-SLP-76. (Lower Right) Anti-SKAP55. (Left) Anti-SKAP55 and anti-SLP-76. (Scale bars correspond to 10 μm.)