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Molecular and Cellular Biology, December 2001, p. 8414-8427, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8414-8427.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

Marie W. Wooten,^1,* Michel L. Vandenplas,^1, M. Lamar Seibenhener,¹ Thangiah Geetha,¹ and Maria T. Diaz-Meco²

Department of Biological Sciences, Auburn University, Auburn, Alabama 36849,¹ and Centro de Biologia Molecular `Severo Ochoa' CSIC, Universidad Autonoma, Canto Blanco, 29049 Madrid, Spain²

Received 18 April 2001/Returned for modification 25 May 2001/Accepted 21 September 2001

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC- becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC- were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC- in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC- were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-. Recruitment of PKC- into the complex was dependent on the tyrosine phosphorylation state of PKC-. The association of src and PKC- was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC- (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-, whereas the Y325F mutation significantly reduced src-induced activation of PKC-. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-B, with significant impairment by the Y325F PKC- mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.

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^* Corresponding author. Mailing address: Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL 36849. Phone: (334) 844-9245. Fax: (334) 844-9234. E-mail: mwwooten{at}acesag.auburn.edu.

Present address: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Molecular and Cellular Biology, December 2001, p. 8414-8427, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8414-8427.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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