Analysis of Cancer‐associated Mutations in the Novel PKC Theta

Abstract

The diacylglycerol‐regulated novel protein kinase C theta is highly druggable, but how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Cancer‐associated mutations in Ca2+‐regulated (conventional) PKC isozymes are generally loss‐of‐function, reframing PKC as having a tumor suppressive property, but whether this is the case for PKC Theta (θ) has not been addressed. Here we examine several cancer‐associated mutations in PKCθ, focusing on those at domain interfaces that are likely to impair autoinhibition, to begin to explore whether this isozyme of the PKC family functions to promote or suppress oncogenic signaling. PKCθ is selectively expressed in hematopoietic cells, namely platelets and T lymphocytes, where it regulates inflammatory signaling. Unsurprisingly, deregulated PKCθ has been implicated in many diseases including cancer where dozens of mutations have already been identified. One such residue was R145 in the autoinhibitory pseudosubstrate segment of PKCθ, whose mutation to His or Cys has been reported for two different cancers. This Arg interacts with Asp465 and Asp508 in the substrate binding cavity to maintain effective autoinhibition in the absence of the allosteric activator, diacylglycerol. We determined the effect of R145H and R145C mutation on the cellular activity and stability of PKCθ. Specifically, we used a genetically‐encoded FRET‐based reporter, C kinase activity reporter 2 (CKAR2) to measure the basal and agonist‐evoked activity of PKCθ. Whereas wild‐type PKCθ was effectively autoinhibited and required addition of agonist (here the phorbol ester, PDBu) for activation, both the R145H and R145C mutants were almost maximally activated under basal conditions. However, addition of cycloheximide to inhibit protein synthesis revealed that the half‐time of turn‐over of the mutants was 3‐ and 8‐fold faster than that of wild‐type enzyme, respectively. Taken together, these data suggest that the R145H and R145C mutants fail to autoinhibit resulting in a PKC with constitutive activity that is unstable and subject to the quality‐control degradation we have previously reported for Ca2+‐regulated PKC isozymes. Thus, these pseudosubstrate mutations in PKCθ are paradoxically loss‐of‐function because the constitutively active kinases are sensitive to degradation.