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J. Biol. Chem., Vol. 278, Issue 26, 24072-24077, June 27, 2003

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Functions of the Activation Loop in Csk Protein-tyrosine Kinase^*

Xiaofeng Lin, Sungsoo Lee and Gongqin Sun 

From the Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881

Autophosphorylation in the activation loop is a common mechanism regulating the activities of protein-tyrosine kinases (PTKs). PTKs in the Csk family, Csk and Chk, are rare exceptions for lacking Tyr residues in this loop. We probed the function of this loop in Csk by extensive site-specific mutagenesis and kinetic studies using physiological and artificial substrates. These studies led to several surprising conclusions. First, specific residues in Csk activation loop had little discernable functions in phosphorylation of its physiological substrate Src, as Ala scanning and loop replacement mutations decreased Csk activity toward Src less than 40%. Second, some activation loop mutants, such as a single residue deletion or replacing all residues with Gly, exhibited 1–2% of wild type (wt) activity toward artificial substrates, but significantly higher activity toward Src. Third, introduction of a thrombin cleavage site to the activation loop also resulted in loss of 98% of wt activity for poly(E₄Y) and loss of 95% of wt activity toward Src, but digestion with thrombin to cut the activation loop, resulted in full recovery of wt activity toward both substrates. This suggested that the catalytic machinery is fully functional without the activation loop, implying an inhibitory role by the activation loop as a regulatory structure. Fourth, Arg³¹³, although universally conserved in protein kinases, and essential for the activity of other PTKs so far tested, is not important for Csk activity. These findings provide new perspectives for understanding autophosphorylation as a regulatory mechanism and imply key differences in Csk recognition of artificial and physiological substrates.

Received for publication, October 16, 2002 , and in revised form, February 21, 2003.

^* This work was supported by Grant 1 P20 RR16457 from the National Institutes of Health and by a grant from the University of Rhode Island Research Council. DNA sequencing was performed at the M.D. Anderson Cancer Center Core Sequencing Facility (supported by National Institutes of Health Grant CA16672). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, 117 Morrill Science Bldg., 45 Lower College Rd., University of Rhode Island, Kingston, RI 02881. Tel.: 401-874-5937; Fax: 401-874-2202; E-mail: gsun{at}uri.edu.

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