Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCtheta in T lymphocytes.
Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.
Thuille
Nikolaus
N
Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria.
Heit
Isabelle
I
Fresser
Friedrich
F
Krumböck
Nina
N
Bauer
Birgit
B
Leuthaeusser
Sabine
S
Dammeier
Sascha
S
Graham
Caroline
C
Copeland
Terry D
TD
Shaw
Steve
S
Baier
Gottfried
G
D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism*; D015533:Transcriptional Activation
D007376:Interleukin-2; D007527:Isoenzymes; D013912:Threonine; D051057:Proto-Oncogene Proteins c-akt; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta