Matthews, Sharon Anne; (1999) Mechanisms controlling the activity and subcellular localisation of PKD/PKCμ. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Protein kinase C (PKC) enzymes have been implicated in a wide range of biological responses in vivo, including cell morphology, differentiation and proliferation. A related kinase is Protein kinase D (PKD), also known as PKCμ. The catalytic domain of PKD is distinct from that of PKC enzymes, although it contains a conserved C1 domain that binds phorbol esters and diacylglycerol (DAG) in a manner similar to classical and novel PKCs. The experiments described here demonstrate that the pharmacological agent bryostatin 1, like phorbol esters, stimulates PKD activity through a novel PKC-dependent signalling pathway in fibroblasts. Importantly, physiological antigen receptor triggering also induces PKC-dependent activation of PKD, in T and B cell lines, in mast cells and in peripheral blood-derived T lymphocytes. Significantly, PKD activity is dynamically regulated by both positive and negative signals from antigen receptors in B lymphocytes. The second cysteine-rich motif within the C1 domain of PKD is the major binding site for phorbol esters, both in vitro and in vivo. However, direct phorbol ester/DAG binding to PKD is dispensable for its activation, as shown by analysis of a PKD C1 domain mutant, consistent with the proposed role for classical and novel PKC enzymes in the regulation of PKD activity. A functional C1 domain is however required for the translocation of PKD from the cytosol to the plasma membrane of intact cells in response to antigen receptor ligation or phorbol ester treatment. Strikingly, PKD only briefly translocates to the plasma membrane of B lymphocytes and mast cells following receptor stimulation, before returning to the cytosol within minutes of the initiation of antigen receptor signalling. In contrast, a sustained phase of PKD activity is observed in antigen receptor-activated B lymphocytes which is maintained over a period of hours. These data suggests that both direct and indirect DAG signalling pathways contribute to the regulation of PKD in vivo and indicate that PKD may disseminate DAG signals away from the plasma membrane into the cell interior during sustained responses to antigen receptor engagement.

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