The regulation of protein kinase C (PKC)ζ in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCζ. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCζ Δ PSS), but mutants designed to mimic phosphorylated PKCζ had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCζ. The T560A mutant also protected PKCζ against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCζ Δ PSS, T410E, PKCζ Δ PSS T410/560E, PKCζ and T560A. Expressed PKCζ activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCζ activity (PKCζ Δ PSS, PKCζ Δ PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation.
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Research Article|
February 15 2004
Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation
J. Ann LE GOOD;
J. Ann LE GOOD
*
Signal Transduction Research Group and Department of Biochemistry, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada
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David N. BRINDLEY
David N. BRINDLEY
1
Signal Transduction Research Group and Department of Biochemistry, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada
1To whom correspondence should be addressed (e-mail david.brindley@ualberta.ca).
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Publisher: Portland Press Ltd
Received:
August 07 2003
Revision Received:
October 23 2003
Accepted:
October 27 2003
Accepted Manuscript online:
October 27 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (1): 83–92.
Article history
Received:
August 07 2003
Revision Received:
October 23 2003
Accepted:
October 27 2003
Accepted Manuscript online:
October 27 2003
Citation
J. Ann LE GOOD, David N. BRINDLEY; Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation. Biochem J 15 February 2004; 378 (1): 83–92. doi: https://doi.org/10.1042/bj20031194
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