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J. Biol. Chem., Vol. 280, Issue 15, 15013-15019, April 15, 2005

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A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D^*

Heike Döppler, Peter Storz, Jing Li¶, Michael J. Comb¶, and Alex Toker||

From the Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and ^¶Cell Signaling Technology, Beverly, Massachusetts 01915

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser⁸² in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.

Received for publication, December 13, 2004 , and in revised form, February 8, 2005.

^* This work was supported by National Institutes of Health Grant CA75134 (to A. T.). 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., RN-237, Boston, MA 02215. Tel.: 617-667-8535; Fax: 617-667-3616; E-mail: atoker{at}bidmc.harvard.edu.

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