The evolutionarily conserved arrangement of domains in SRC family kinases is important for substrate recognition

Abstract

The SH3-SH2-kinase domain arrangement in nonreceptor tyrosine kinases has been conserved throughout evolution. For Src family kinases, the relative positions of the domains are important for enzyme regulation; they permit the assembly of Src kinases into autoinhibited conformations. The SH3 and SH2 domains of Src family kinases have an additional role in determining the substrate specificity of the kinase. We addressed the question of whether the domain arrangement of Src family kinases has a role in substrate specificity by producing mutants with alternative arrangements. Our results suggest that changes in the positions of domains can lead to specific changes in the phosphorylation of Sam68 and Cas by Src. Phosphorylation of Cas by several mutants triggers downstream signaling leading to cell migration. The placement of the SH2 domain with respect to the catalytic domain of Src appears to be especially important for proper substrate recognition, while the placement of the SH3 domain is more flexible. The results suggest that the involvement of the SH3 and SH2 domains in substrate recognition is one reason for the strict conservation of the SH3-SH2-kinase architecture.

Figure 1

Domain arrangements in c-Src and mutants. The mutants are named according to the position of the domains from the N- to C-terminus. The unique domain is colored white, the SH3 domain yellow, the SH2 domain green, and the catalytic domain blue. The position of the Tyr527 sequence (normally present in the C-terminal tail of wild-type Src) is indicated. The R175L/W118A construct contains point mutations that block ligand binding to the SH2 and SH3 domains. The amino acid sequences of the constructs are given in Figure 1 of the Supporting Information.

Figure 2

Phosphorylation of c-Src and the mutants. (A) SYF cells were transiently transfected with wild-type c-Src or the mutants. The names of the constructs are given in Figure 1: the unique domain is denoted by U, the SH3 domain by 3, the SH2 domain by 2, and the kinase catalytic domain by C. The cells were harvested 40 h post-transfection, and whole cell lysates (10 μg) were separated via 10% SDS–PAGE and transferred onto PVDF membranes. The membranes were probed with anti-pY416 antibody. To assess expression, membranes were probed with anti-Flag antibody. Anti-tubulin antibody was included as a loading control. Vector represents SYF cells transfected with empty vector alone. The figure is representative of three separate experiments; a comparison of results from three different transfected cell lysates is shown in Figure 2 of the Supporting Information. (B) Transfected SYF cells were lysed and subjected to an immunoprecipitation reaction with anti-Flag antibody. The immunoprecipitates were separated by 10% SDS–PAGE, transferred to a PVDF membrane, and analyzed by anti-pY527 Western blotting.

Figure 3

Kinase activity of immunoprecipitated proteins. (A) SYF cells expressing wild-type Src or the mutants were lysed in RIPA buffer. Kinases were isolated by immunoprecipitation with Flag-agarose beads, and the samples were divided into three equal parts. Two samples were used in duplicate kinase assays with [γ-³²P]ATP and the substrate peptide AEEEIYGEFEAKKKKG (top). One sample was analyzed by anti-Flag Western blotting to compare the amounts of kinases (bottom). (B) SYF cells were transiently transfected with the indicated constructs. Kinases were isolated by immunoprecipitation with Flag-agarose beads and assayed as described for panel A. The inset shows IP samples were analyzed by anti-Flag Western blotting.

Figure 4

Global tyrosine phosphorylation analysis of Src and mutants. SYF cells were transiently transfected with c-Src or mutants. Cells were harvested 40 h post-transfection, and lysates (50 μg) were separated via 10% SDS–PAGE and transferred onto a PVDF membrane. The membrane was probed with anti-pTyr antibody and reprobed with anti-Flag antibody to check expression. The membrane was probed with anti-tubulin antibody as a loading control. Vector represents SYF cells transfected with empty vector alone. The figure is representative of three separate experiments.

Figure 5

Phosphorylation of Cas by Src and the mutants. (A) SYF cells were transiently transfected with wild-type Src or the mutants. The cells were harvested 40 h post-transfection, and lysates were subjected to immunoprecipitation using anti-Cas antibody. The immunoprecipitates were separated via 7.5% SDS–PAGE, and Western blotting was carried out with anti-pTyr antibody. The membrane was stripped and reprobed with anti-Cas antibody to ensure equivalent Cas immunoprecipitation. The figure is representative of three separate experiments; a comparison of results from three different transfected cell lysates is shown in Figure 3 of the Supporting Information. (B) SYF cells expressing WT Src, mutant 3U2C, or vector control were lysed and subjected to immunoprecipitation reactions using anti-Flag antibody. Purified Cas (WT or F17-Cas) was added to the immunoprecipitates, and kinase reactions proceeded for 30 min at 30 °C. Reactions were stopped with SDS sample buffer, and mixtures were analyzed by anti-phosphotyrosine Western blotting. The membranes were stripped and reprobed with anti-Cas and anti-Flag antibodies.

Figure 6

Wound healing assays. In the top panel, SYF cells were infected with retroviruses expressing c-Src or the domain-rearranged mutants. The cells were more than 90% infected as measured by GFP fluorescence (data not shown). Multiple wounds were scratched on the 60 mm dishes, and eight positions were photographed for 10–12 h. The rate of wound closure was calculated by plotting the width of the wound vs time. The rate of closure shown above is the average of eight separate positions. The standard errors are those for all eight positions. In the bottom panel, expression of the constructs in SYF cells was tested by anti-Flag Western blotting.

Figure 7

Phosphorylation of Sam68 by Src and the mutants. SYF cells were transiently transfected with wild-type Src or the mutants and arrested in mitosis using nocodazole 24 h after transfection. The mitotic cells were harvested 40 h post-transfection, and lysates were subjected to immunoprecipitation using anti-Sam68 antibody. The immunoprecipitates were separated by 10% SDS–PAGE, and Western blotting was carried out with anti-pTyr antibody. The membranes were stripped and reprobed with anti-Sam68 antibody to ensure equivalent Sam68 immunoprecipitation. The figure is a representative of three separate experiments; a comparison of results from three different transfected cell lysates is shown in Figure 4 of the Supporting Information.

Figure 8

In vitro kinase assays using substrates with SH3 ligands. Kinase assays were performed using purified proteins and the indicated peptides at 50 μM (sequences given in Table 2). Initial rates were measured in triplicate, and they are plotted relative to the control substrate ± the standard deviation.

Figure 9

In vitro kinase assays using substrates with SH2 ligands. Kinase assays were performed using purified proteins and the indicated peptides at 50 μM (sequences given in Table 2). Initial rates were measured in triplicate. The rates of phosphorylation of substrate-pYEEI and pYEEI-substrate are presented relative to the appropriate control peptide.