PMC

Structure of Cas. The SH3 domain, substrate domain and Src interaction sites are indicated.

Structure of attenuated Src kinase domain/Cas chimeras. The Src/Cas(SD) chimeras were constructed as described in the materials and methods. Src* is the isolated attenuated Src kinase domain (Y416F). Src*/Cas(SD) is a fusion of the attenuated Src kinase domain to a.a. L¹⁵⁷ to R⁵¹⁶ of the Cas substrate domain. Src*/Cas(5'SD) is a fusion of the attenuated Src kinase domain to the 5' portion of the Cas substrate domain (a.a. L¹⁵⁷ to A³²⁴). Src*/Cas(3'SD) is a fusion of the attenuated Src kinase domain to the 3' portion of the Cas substrate domain (a.a. S³²⁵ to R⁵¹⁶). Src^KM is the isolated inactive kinase domain (K295M). Src^KM/Cas(5'SD) is a fusion of the inactive Src kinase domain to the 5' portion of the Cas substrate domain (a.a. L¹⁵⁷ to R⁵¹⁶). Src^KM/Cas(3'SD) is a fusion of the inactive Src kinase domain to the 3' portion of the Cas substrate domain (a. a. S³²⁵ to R⁵¹⁶).

The Src*/Cas(SD) chimera is highly tyrosine phosphorylated when expressed in cells and associates with tyrosine phosphorylated proteins. a) Cos-7 cells were transfected with vector alone (lane 1), plasmid coding for the isolated attenuated Src kinase domain (lane 2) or the Src*/Cas(SD) chimera (lane 3). Cell lysates were probed with antibodies to phosphotyrosine. The arrow indicates the Src*/Cas(SD) chimera. b) Cos-7 cells were transfected with vector alone (lane 1) or plasmid coding for the Src*/Cas(SD) chimera (lane 2). The Src*/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the Src*/Cas(SD) chimera (bottom panel). The arrow indicates the Src*/Cas(SD) chimera.

The tyrosine phosphorylated Cas substrate domain competes with endogenous Cas for binding to v- and c-crk. a) Cos-7 cells were transfected with vector alone (lane 1), the Src*/Cas(3'SD) chimera alone (lanes 2 and 3), v-crk alone (lane 4) or the Src*/Cas(3'SD) chimera along with v-crk (lane 5). The Src*/Cas(3'SD) chimera or v-crk was then immunoprecipitated and probed for phosphotyrosine. Note that v-crk normally associates with more Cas when transfected alone compared when transfected with the Src*/Cas(3'SD) chimera. b) Cos-7 cells were transfected with vector (lane 1) or the Src*/Cas(SD) chimera (lane 2). Endogenous c-crk was then immunoprecipitated and probed with anti-phosphotyrosine (top panel) or with anti-c-crk (bottom panel).

Tyrosine phosphorylated substrate domain of Cas blocks v-crk mediated transformation. a) NIH3T3 cells or those transformed with v-crk were either transfected with vector alone or with vector carrying the Src*/Cas(SD) chimera. Whole cell extracts (WCL) from cells were probed with anti-phosphotyrosine (top panel) or with anti-HA antibody (bottom panel). b) NIH3T3 cells stably transfected with vector alone (left panel), with v-crk (middle panel) or v-crk and the Src*/Cas(SD) chimera were assayed for growth in soft agar. Representative sections are shown. Colony numbers per view field were as follows: 26.1 (+/- 4.2) for v-crk alone vs. 2.9 (+/- 1.4), statistically significant (p = 7.76 × 10^-8, t-test).

Tyrosine phosphorylated Cas substrate domain affects c-crk interaction with C3G, but not endogenous FAK or Cas tyrosine phosphorylation.a) Lysates from NIH3T3 cells stably carrying vector alone or expressing the Src*/Cas(SD) chimera were immunoprecipitated with anti-phosphotyrosine (top panel), or anti-crk (middle and bottom panels) and probed with anti-HA to detect the Src*/Cas(SD) chimera (top and middle panels), or C3G (bottom panel). Lanes 1 and 2, vector and the Src*/Cas(SD) chimera transfected NIH3T3 cells respectively, whole cell extract; lanes 3 and 4, vector transfected NIH3T3 cells immunoprecipitated with anti-phosphotyrosine antibody; lane 5, NIH3T3 cells expressing the Src*/Cas(SD) chimera immunoprecipitated with anti-phosphotyrosine antibody. Arrows indicate the Src*/Cas(SD) chimera and C3G in the top, middle and bottom panels. b) A duplicate blot to that shown in (a) was probed with anti-Fak antibodies (top panel) or with anti-Cas antibodies (bottom panel). Arrows indicate FAK and Cas.

Tyrosine phosphorylated Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).

v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (Src^KM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the Src^KM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The Src^KM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the Src^KM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the Src^KM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. Src^KM/Cas(5'SD) chimera (lane 1), Src^KM/Cas(3'SD) chimera (lane 2), and Src^KM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).