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J. Biol. Chem., Vol. 281, Issue 41, 30947-30956, October 13, 2006

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Targeting Protein Kinase C Activity Reporter to Discrete Intracellular Regions Reveals Spatiotemporal Differences in Agonist-dependent Signaling^*

Lisa L. Gallegos, Maya T. Kunkel, and Alexandra C. Newton¹

From the Department of Pharmacology and the Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92093

Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH₂ or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca²⁺ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca²⁺, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.

Received for publication, April 19, 2006 , and in revised form, August 8, 2006.

^* This work was supported by National Institutes of Health Grants P01 DK54441 and GM-43154. 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: Dept. of Pharmacology, University of California at San Diego, 9500 Gilman Dr. 0721, La Jolla, CA 92039-0721. Tel.: 858-534-4527; Fax: 858-822-5888; E-mail: anewton{at}ucsd.edu.

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