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Autophosphorylation of Src and Yes Blocks Their Inactivation by Csk Phosphorylation

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Autophosphorylation of Src and Yes Blocks Their Inactivation by Csk Phosphorylation Oncogene (1998) 17, 1587 ± 1595 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation Gongqin Sun, Ajay K Sharma and Raymond JA Budde Department of Neuro-Oncology, Box 316, University of Texas, MD Anderson Cancer Center, Houston, Texas 77030, USA Csk phosphorylates Src family protein tyrosine kinases proto-oncogenes, since their activation by mutation on a tyrosine residue near their C-terminus and down- leads to transformation of the host cells (Courtneidge, regulates their activity. We previously observed that this 1994). Elevation of the enzymatic activity of some regulation requires a stoichiometric ratio of Csk : Src in members has been associated with many types of a time-independent manner. In this report we examined human cancer (Levitzki, 1996). In tumor cells where this unusual kinetic behavior and found it to be caused by Src activity is elevated, the increase in its activity is Src autophosphorylation. First, pre-incubation of Src mostly the result of increased speci®c activity (Bolen et with ATP-Mg led to time-dependent autophosphoryla- al., 1987; Talamonti et al., 1993). Furthermore, no tion of Src, activation of its kinase activity and loss of its mutation has been identi®ed in humans that is ability to be inactivated by Csk. However, the autopho- responsible for the increased speci®c activity. These sphorylated Src can still be phosphorylated by Csk. The ®ndings suggest that elevation of Src activity in human SH2 binding site for phospho-Tyr of this hyperactive and tumor cells is due to a disruption of its regulation. For doubly phosphorylated form of Src is not accessible. this reason, understanding the regulation of Src family Second, dephosphorylation of autophosphorylated Src by PTKs may be one of the keys in understanding and protein tyrosine phosphatase 1B allowed Src to be designing eective treatment for cancers that have inactivated by Csk. Third, protein tyrosine phosphatase elevated activity of Src family PTKs. 1B preferentially dephosphorylates the Src autopho- A variety of stimuli, such as various growth factors, sphorylation site and allows for Src regulation by Csk. UV radiation and oxidative stress can directly or Finally, Yes, another member of the Src family, was also indirectly modulate Src activity. The ability to respond only partially inactivated when a sub-stoichiometric to these signals depends on a complex multi-domain amount of Csk was used. Mutation of the tyrosine structure. All PTKs in the Src family have an N- autophosphorylation site of Yes to a phenylalanine terminal acylation motif that is required for their resulted in a mutant Yes enzyme that can be fully membrane association, a unique region that is inactivated by a sub-stoichiometric amount of Csk in a heterologous in the family, two Src homology time-dependent manner. These results demonstrate that domains (SH3 and SH2) that mediate protein-protein Csk phosphorylation inactivates Src and Yes only when interactions, an SH1 catalytic domain which carries out they are not previously autophosphorylated and Src the phosphorylation reaction, and a regulatory Tyr (Yr) autophosphorylation can block the inactivation by Csk phosphorylation motif located at the C-terminal phosphorylation. This conclusion suggests a dynamic region. Another ubiquitous feature among Src family model for the regulation of the Src family protein PTKs is the presence of a Tyr autophosphorylation site tyrosine kinases, which is discussed in the context of (Ya) within the activation loop located in the catalytic previously reported observations on the regulation of Src domain (Smith et al., 1993). At the molecular level, family protein tyrosine kinases. several mechanisms that modulate Src activity have been discovered, including binding to other proteins Keywords: Src and Yes regulation; Csk; protein through the SH2 and SH3 domains, phosphorylation tyrosine phosphatase 1B; autophosphorylation of multiple Ser/Thr residues, Tyr phosphorylation by receptor PTKs, autophosphorylation and Csk phos- phorylation (Brown and Cooper, 1996). The major regulatory mechanism is apparently phosphorylation of Introduction Yr by Csk, which results in inactivation of Src (Cooper et al., 1986). In csk knockout mice, Src is mostly Protein tyrosine kinases (PTKs) of the Src family play unphosphorylated on the C-terminal regulatory Tyr important roles in cellular signal transduction (Brickell, and has elevated kinase activity (Imamoto and 1992). There are nine members: Src, Yes, Fyn, Yrk, Soriano, 1993; Nada et al., 1993). Src expressed in Fgr, Hck, Lyn, Lck and Blk. Src, Yes and Fyn are yeast is minimally phosphorylated on Yr and highly expressed ubiquitously, while the other six are active, while co-expression of Csk led to phosphoryla- expressed in restricted cell types, particularly in tion of Yr and inactivation of Src (Superti-Furga et al., haematopoietic cells. These PTKs are key switches in 1993). These ®ndings establish Csk as the negative regulating many cellular signal transduction pathways regulator of Src family PTKs. (Thomas and Brugge, 1997). Most of these enzymes are In this communication, we examined the kinetics of Src inactivation by Csk and found that autopho- sphorylation of Src and Yes blocks their inactivation but not phosphorylation by Csk. This ®nding indicates Correspondence: RJA Budde Received 12 February 1998; revised 23 April 1998; accepted 23 April that autophosphorylation plays a greater role in 1998 regulating Src and Yes activity than previously Regulation of Src, Yes by auto- and trans-phosphorylation GSunet al 1588 understood and suggests that an altered level of required a stoichiometric amount of Csk to achieve autophosphorylation may be the reason for the maximal inactivation of Src. Second, prior phosphor- paradoxically activated Src in malignant human cell ylation by Csk (pre-incubation with Csk and ATP-Mg) lines with high levels of active Csk. does not lead to signi®cantly higher levels of inactivation of Src than without prior phosphorylation by Csk (incubation with no MgCl2). This apparent time Results independence is further demonstrated in Figure 1b. At the selected Csk to Src ratios of 1 : 5 and 1 : 2, In order to better understand the regulation of Src by approximately 30% and 60% of Src was inactivated Csk and identify factors that may aect this regulation, and this was independent of the pre-incubation time. we reconstituted this system in vitro with puri®ed Incubation of Src with ATP-MG in the absence of Csk recombinant enzymes. In the process, we observed that (Csk to Src ratio of 0 : 1) resulted in activation of Src the inactivation of Src by Csk does not follow a as a result of its autophosphorylation. These observa- normal enzymatically catalyzed process. Such observa- tions were not a function of the particular enzyme tions are demonstrated by pre-incubating Src with ATP samples used or methods of their expression and and dierent amounts of Csk in the presence or isolation. Data consistent with these observations absence of MgCl2 for 30 min before the Src activity have been reported repeatedly in the literature with was assayed with carboxymethylated-maleylated, re- dierent members of the Src family (Okada and duced lysozyme (RCM-L) (Figure 1a). In both Nakagawa, 1989; Ruzzene et al., 1994; Koegl et al., treatments, Src activity decreased with the increase in 1994) and Csk from dierent sources. Such consistency the amount of Csk added to the assay. While these suggested that these observations are a re¯ection of the results con®rmed that Csk was able to inactivate Src, intrinsic properties of Src regulation by Csk phosphor- two points in these results were unexpected. First, it ylation. These observations suggested that Src inactivation by Csk phosphorylation is not a simple ATP-Mg- dependent enzymatic conversion of active to inactive Src. There appears to be other factor(s) aecting this process. There are three possibilities that are consistent with these observations: (1) Csk inactivation of Src requires the formation of a stable Src-Csk complex after phosphorylation; (2) Csk is quickly inactivated by incubation with Src and/or ATP-Mg and thus unable to inactivate additional molecules of Src; and (3) Src is quickly transformed upon incubation with Csk and/or ATP-Mg into a form that can not be inactivated by Csk. The ®rst possibility would be consistent with the high stoichiometry of Csk required and the lack of time-dependent inactivation of Src. Furthermore, it was reported that Csk can form a complex with members of the Src family (Ruzzene et al., 1994; Bougeret et al., 1996). However, evidence from the literature and our own results eliminated this possibility. First, attempts by others (Sabe et al., 1992) and us (data not shown) have failed to detect strong and stable complexes between Csk and Src. Second, Src isolated after pre-incubation with Csk and ATP-Mg remain inactivated (Stover et al., 1994). Third, the ability of Csk deletion mutants (deletions in SH2 or SH3 domains) to inactivate Src correlates with the kinase activity of the mutants (data not shown). These results indicate that Src inactivation by Csk is a phosphorylation dependent process and does not require the formation of a stable Csk-Src complex. We also examined and eliminated the possibility of Csk being inactivated by incubation with Src and/or ATP- Mg. Csk undergoes autophosphorylation to a low Figure 1 Unusual kinetic behaviors of Src inactivation by Csk. extent (Amrein et al., 1995) and this does not (a) Dosage-dependent inactivation of Src by Csk. Src signi®cantly aect its activity (Oetken et al., 1994; 71 (0.13 mgml ) was pre-incubated for 30 min at 308C with ATP Sun et al., 1997). Furthermore, Csk is neither and dierent amount of Csk in the presence or absence of MgCl2 (10 mM). After pre-incubation, Src activity was assayed with phosphorylated nor inactivated by Src even when a RCM-lysozyme as the substrate.
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