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J. Biol. Chem., Vol. 278, Issue 39, 38015-38021, September 26, 2003

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Atypical Protein Kinase C Plays a Critical Role in Protein Transport from Pre-Golgi Intermediates^*

Ellen J. Tisdale , Jing Wang, Robert B. Silver and Cristina R. Artalejo

From the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201

The small GTPase Rab2 requires atypical protein kinase C / (PKC/) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702–712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKC/ in the early secretory pathway. A peptide corresponding to the unique PKC/ pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKC/, -COP, and p53/p58. Because Rab2, -COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKC/ (W274K). Both the pseudosubstrate peptide and kinase-dead PKC/ in tandem with Rab2 caused sustained membrane association of PKC/, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKC/ was reversed by PKC/ wild type. These combined results indicate that PKC/ is essential for protein transport in the early secretory pathway and suggest that PKC/ kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

Received for publication, May 22, 2003 , and in revised form, July 12, 2003.

^* This work was supported by American Heart Association Midwest Affiliate Grant 0030385Z (to E. J. T.), Public Health Service Grant DK58921 (to C. R. A.), and NSF, National Institutes of Health Grant IBN-9985874 (to C. R. A.). 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 Pharmacology, Wayne State University School of Medicine, 6374 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1007; Fax: 313-577-6739; E-mail: etisdale{at}med.wayne.edu.

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