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J. Biol. Chem., Vol. 282, Issue 42, 31094-31102, October 19, 2007

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S-Adenosylmethionine-dependent Protein Methylation in Mammalian Cytosol via Tyrphostin Modification by Catechol-O-methyltransferase^*

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095

It has previously been shown that incubation of mammalian cell cytosolic extracts with the protein kinase inhibitor tyrphostin A25 results in enhanced transfer of methyl groups from S-adenosyl-[methyl-³H]methionine to proteins. These findings were interpreted as demonstrating tyrphostin stimulation of a novel type of protein carboxyl methyltransferase. We find here, however, that tyrphostin A25 addition to mouse heart cytosol incubated with S-adenosyl-[methyl-³H]methionine or S-adenosyl-[methyl-¹⁴C]methionine stimulates the labeling of small molecules in addition to proteins. Base treatment of both protein and small molecule fractions releases volatile radioactivity, suggesting labile ester-like linkages of the labeled methyl group. Production of both the base-volatile product and labeled protein occurs with tyrphostins A25, A47, and A51, but not with thirteen other tyrphostin family members. These active tyrphostins all contain a catechol moiety and are good substrates for recombinant and endogenous catechol-O-methyltransferase. Inhibition of catechol-O-methyltransferase activity with tyrphostin AG1288 prevents both base-volatile product formation and protein labeling from methyl-labeled S-adenosylmethionine in heart, kidney, and liver, but not in testes or brain extracts. These results suggest that the incorporation of methyl groups into protein follows a complex pathway initiated by the methylation of select tyrphostins by endogenous catechol-O-methyltransferase. We suggest that the methylated tyrphostins are further modified in the cell extract and covalently attached to cellular proteins. The presence of endogenous catechols in cells suggests that similar reactions can also occur in vivo.

Received for publication, July 3, 2007 , and in revised form, August 14, 2007.

^* This work was supported by National Institutes of Health Grant GM026020 (to S. G. C.) and by USPHS Training Program Grant GM07185 (to R. S. L.). 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.

¹ To whom correspondence should be addressed: UCLA Dept. of Chemistry and Biochemistry and the Molecular Biology Institute, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569. Tel.: 310-825-8754; Fax: 310-825-1968; E-mail: clarke{at}mbi.ucla.edu.

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