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Published online 3 January 2002. doi:10.1083/jcb.200110047
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© The Rockefeller University Press, 0021-9525/2002/1/65 $5.00
The Journal of Cell Biology, Volume 156, Number 1, January 7, 2002 65-74

Article

 Structural requirements for localization and activation of protein kinase C µ (PKCµ) at the Golgi compartment

Angelika Hausser1, Gisela Link1, Linda Bamberg1, Annett Burzlaff1, Sylke Lutz1, Klaus Pfizenmaier1 and Franz-Josef Johannes2

1 Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany
2 Fraunhofer Institute for Interfacial Engineering, 70569 Stuttgart, Germany

Address correspondence to Dr. F.J. Johannes, Institute of Cell Biology and Immunology, Allmandring 31, 70569 Stuttgart, Germany. Tel.: (49) 711685-6995. Fax: (49) 711685-7484. E-mail: fjj{at}igb.fhg.de

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC)µ involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKCµ–green fluorescent protein fusion proteins costained with the Golgi compartment–specific markers p24 and p230. Deletions of either the NH2-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKCµ completely abrogated Golgi localization of PKCµ. As an NH2-terminal PKCµ fragment was colocalized with p24, this region of PKCµ is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKCµ to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKCµ found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKCµ, in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH2-terminal serine(s) in the regulatory domain. PKCµ activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKCµ function at the Golgi compartment.

Key Words: PKCµ; Golgi localization; activation; phosphorylation; green fluorescent protein


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