Membrane targeting and cytoplasmic sequestration in the spatiotemporal localization of human protein kinase C alpha

In order to map the molecular determinants that dictate the subcellular loc alization of human protein kinase C alpha (hPKC alpha), full-length and del etion mutants of hPKC alpha were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotro pin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKC alpha-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKC alpha failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracell ular calcium concentration in these cells. TRH-stimulated translocation of hPKC alpha-GFP from the cytoplasm to cell-cell contacts was a biphasic proc ess: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the transloc ation induced by phorbol 12-myristate 13-acetate absolutely required the N- terminal V1 region. In contrast to the full-length hPKC alpha, the N-termin al regulatory domain alone or associated with the V3 hinge region was spont aneously and uniformly localized at the plasma membrane of single and appos ed cells. However, treatment with the calcium chelator BAPTA/AM induced a d ifferential cytoplasmic/nuclear redistribution of the regulatory domain, de pending on its association with V3, which suggests the existence of a mecha nism controlling the cytoplasmic sequestration of inactive hPKC alpha and i nvolving the V3 region. By using other deletion mutants, we were able to ma p the sequence required for this sequestration to the C2+V3 regions. This w ork points to the existence of a complex interplay between membrane targeti ng and cytoplasmic sequestration in the control of the spatiotemporal local ization of hPKC alpha.