Phosphorylation of Thr642 is an early event in the processing of newly synthesized protein kinase C beta 1 and is essential for its activation.
Earlier studies of a site-specific mutant of protein kinase C beta 1 (PKC beta 1) altered at Thr635 and Thr642 indicated that these phosphorylation sites are critical for enzymatic function (Zhang, J., Wang, L., Petrin, J., Bishop, W. R., and Bond, R. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,6130-6134). To determine the contribution of the individual threonines, we report here on two site-specific mutants in which either Thr635 or Thr642 was changed to alanine. When transiently overexpressed in Cos cells wild-type PKC beta 1 exists in two forms: a Triton-insoluble form with high electrophoretic mobility and a slower migrating Triton-soluble form. Mutation at Thr642 (but not Thr635) results in production of only the fast-migrating form. [35S]Methionine pulse-chase labeling indicates that wild-type PKC beta 1 is synthesized as the fast-migrating form and is subsequently converted to the slow-migrating form. 32P labeling shows that only the slow-migrating form is a phosphoprotein. Mutation of Thr642 abolishes this phosphorylation. Finally, the Thr642 mutant PKC beta 1 lacks enzymatic activity and, when expressed in NIH 3T3 cells, reduces phorbol ester-induced c-fos promoter activity. These results indicate that Thr642 phosphorylation is an early event in the processing of newly synthesized PKC beta 1 and is required for enzymatic function. These results support a role for a PKC kinase in PKC processing and activation.
Molecular Pharmacology Section, Schering-Plough Research Institute, Kenilworth, New Jersey 07003.
J
J
Zhang
L
L
Wang
J
J
Schwartz
R W
RW
Bond
W R
WR
Bishop
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism
D010750:Phosphoproteins; D011994:Recombinant Proteins; D013912:Threonine; D011493:Protein Kinase C; D064546:Protein Kinase C beta