The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction.
The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae. Swapping the regulatory domains between SRA and PRC revealed that N117D, I96T, and V124L mutations in the n-src- and RT-loops of the SH3 domain of PRC are responsible for the low kinase activity of PRC. Moreover, introducing D117N, R95W, T96I, and L124V into activated c-Src(Y527F) caused a 2.5-fold increase in its activity. The mutations in the CD linker KP249,250DG and L255A, which were shown to activate c-Src, had no effect on the activity of the "SH2-activated" Src kinases. Together our data suggest that in the "SH2-activated" forms of Src, the SH3 domain continues to influence the kinase activity via the direct contacts of the n-src- and RT-loops with the kinase N-terminal lobe.
Brábek
Jan
J
Department of Physiology and Developmental Biology, Charles University, Vinicna 7, 128 00 Prague 2, Czech Republic.
Mojzita
Dominik
D
Novotný
Marian
M
Půta
Frantisek
F
Folk
Petr
P
D000818:Animals; D002645:Chickens; D015536:Down-Regulation; D008958:Models, Molecular; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018909:src Homology Domains
D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)