Role of post-translational modifications on cell distribution and FUNCTIONS of protein kinase C theta

The novel protein kinase C (PKC) theta isozyme has been recently proposed as a drug target for human leukemias. In single cell types we have identified multiple PKC theta forms, characterised by differential post-translational modifications. Aim of this work was to establish if these molecular modifications affect PKC theta intracellular localisation and catalytic competence. A critical role in the control of both PKC theta cell distribution and catalytic activity is played by the phosphorylation state of the kinase activation loop, at the Thr538 residue. PKC theta molecules showing a Mr of 85 kDa (named theta-85) and dephosphorylated at Thr538 have been found specifically associated with the Golgi complex and catalytically active. In contrast, PKC theta molecules with a Mr of 76 kDa (named theta-76), partially phosphorylated at Thr538, are localised in the detergent-soluble cell fraction. Only theta-76 kinase forms not phosphorylated at Thr538 can undergo conversion to theta-85 by an autophosphorylation process. Cell treatment with calyculin A, a protein phosphatase 1 and 2A inhibitor or with LY294002, an inhibitor of Thr538 phosphorylation via PI3K/PDK1, results in a significant increase in the amount of protein kinase PKC theta associated with the Golgi complex. Moreover, as demonstrated by the behaviour of Thr538ÆAla and Thr538 ÆGlu PKC theta mutants, the absence of pThr538 is sufficient for the recruitment of PKC theta to the Golgi complex. Moreover, the Thr538ÆAla PKC theta mutant also results modified by Nglycosylation. This kinase form binds wheat germ agglutinin and acquires a lower Mr by treatment with N-glycosydase F. These findings suggest that different PKC theta forms might be involved in distinct cell functions. New pharmacological strategies, aimed specifically to control PKC theta activities that promote malignant cell proliferation, should be designed and screened on the basis of their effect on individual PKC theta forms.