Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCθ in T lymphocytes

Abstract

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)h. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCh in primary CD3 þ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCh to mediate NF-jB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCh's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCh with Akt/PKBa in NF-jB 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 PKCh in activated T cells. The PKChT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCh at Thr-219 plays an important role in the correct targeting and cellular function of PKCh upon antigen receptor ligation.

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Ser-317 is located in the hinge domain and is not conserved on other [128]. Thr-219 on PKC is also phosphorylated in T lymphocytes in response to TCR stimulation and has been reported to be an autophosphorylation site [97], thereby serving as a useful marker of catalytic activity.…”

“…While phosphorylation at Thr-219 does not affect the catalytic activity of PKC or its ability to bind lipid second messengers, phosphorylation at this site is required for the proper localisation of the enzyme in stimulated T cells [97]. Phosphoproteomics analysis has also revealed that PKC undergoes phosphorylation at Thr-685 between the TM and HM sites in response to TCR-stimulation [101].…”

The Potent Protein Kinase C-Selective Inhibitor AEB071 (Sotrastaurin) Represents a New Class of Immunosuppressive Agents Affecting Early T-Cell Activation

“…PKC was assayed according to Geiges et al (1997). In situ Thr-219 autophosphorylation status analysis of PKC was done by a phospho-site-specific antibody as described in Thuille et al (2005).…”

Response to Comment on "PDK1 Nucleates T Cell Receptor-Induced Signaling Complex for NF-κB Activation"

“…However, our report (2) is not the first to demonstrate inducible phosphorylation of PKCq on Thr-538 in T cells (3)(4)(5). Gruber et al have also reported constitutive phosphorylation in previous publications (6,7). Clearly, investigators are divided on whether Thr-538 phosphorylation is inducible or constitutive, with the caveat that, to date, reports of constitutive phosphorylation have been based mostly on overexpression experiments.…”