The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction q Jan Br abek, 1 Dominik Moj  zita, 1 Marian Novotn y, Franti  sek P  uta, and Petr Folk * Department of Physiology and Developmental Biology, Charles University, Vinicna 7, 128 00 Praha 2, Czech Republic Received 8 July 2002 Abstract The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of in- teractions, 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: v-Src; c-Src; SH3 Domain; Kinase; CD Linker; Saccharomyces cerevisiae Proper regulation of the non-receptor tyrosine kinase c-Src is crucial for the control of the cell cycle or contact mediated growth-inhibition [1]. The phosphorylation by, as well as of, c-Src is of key importance for the signaling through growth factor, integrin or G-protein coupled receptors [1]. The phosphorylation of substrates by c-Src often immediately regulates the phosphoryla- tion status of c-Src itself, e.g., through Csk or PTPs [2]. Changes in c-Src catalytic activity are accompanied by changes in its binding properties, as both c-Src and, often, its substrates possess SH3 and SH2 domains. A complicated network of intramolecular interactions among the SH3, SH2, and catalytic domains and the linker regions of c-Src translates regulatory inputs into catalytic and binding activities (for review see [2]). The protooncogenic c-Src can be permanently acti- vated by frame-shift mutation in the C-terminus, which eliminates the tail’s binding by the SH2 domain and gives rise to the oncoprotein v-Src [3]. This type of ac- tivation was originally discovered to be the underlying principle of the ability of Rous Sarcoma Virus (RSV) to transform cells. We refer in the text to ‘‘SH2-activated’’ or ‘‘SH2-inhibited’’ Src, when the C-terminal tail bind- ing to the SH2 domain is abolished or in effect, respec- tively. The most widely used v-Src variant, derived from the Schmidt-Ruppin A strain of RSV (SRA; [4]), is 5– 25 times more active in vitro than SH2-inhibited c-Src. We found that v-Src derived from the low oncogenic Prague C strain of RSV (PRC; [5]) is 5 times less active than SRA and is thus closer to c-Src, despite the lack of the inhibitory interaction between its SH2 domain and the C-terminus [6]. We were able to ascribe the differ- ences in kinase activity between PRC and SRA to amino acid substitutions in their regulatory domains. The crystal structures of chicken c-Src, human c-Src [7], and the related Hck kinase [8], all in the SH2- inhibited conformation and without the unique do- main, allowed to outline the framework of regulatory Biochemical and Biophysical Research Communications 296 (2002) 664–670 www.academicpress.com BBRC q Abbreviations: PTP, protein tyrosine phosphatase; SH, Src homol- ogy; RSV, Rous sarcoma virus; SRA, v-Src derived from Schmidt- Ruppin A strain of RSV; PRC, v-Src derived from Prague C strain of RSV; FAK, focal adhesion kinase; CD, catalytic domain; SRM, kinase inactive mutant of v-Src derived from Schmidt-Ruppin A. * Corresponding author. Fax: +4202-2195-3242. E-mail address: dicty@mbox.cesnet.cz (P. Folk). 1 These authors contributed equally to the work. 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)00884-7