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 localization of human protein kinase C alpha (hPKCalpha), full-length and deletion mutants of hPKCalpha were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKCalpha-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKCalpha failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracellular calcium concentration in these cells. TRH-stimulated translocation of hPKCalpha-GFP from the cytoplasm to cell-cell contacts was a biphasic process: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the translocation induced by phorbol 12-myristate 13-acetate absolutely required the N-terminal V1 region. In contrast to the full-length hPKCalpha, the N-terminal regulatory domain alone or associated with the V3 hinge region was spontaneously and uniformly localized at the plasma membrane of single and apposed cells. However, treatment with the calcium chelator BAPTA/AM induced a differential cytoplasmic/nuclear redistribution of the regulatory domain, depending on its association with V3, which suggests the existence of a mechanism controlling the cytoplasmic sequestration of inactive hPKCalpha and involving the V3 region. By using other deletion mutants, we were able to map the sequence required for this sequestration to the C2+V3 regions. This work points to the existence of a complex interplay between membrane targeting and cytoplasmic sequestration in the control of the spatiotemporal localization of hPKCalpha.
Vallentin
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A
INSERM U469, 141 rue de la Cardonille, 34094 Montpellier cedex 5, France.
Prévostel
C
C
Fauquier
T
T
Bonnefont
X
X
Joubert
D
D
D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D017353:Gene Deletion; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism*; D018613:Microscopy, Confocal; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013973:Thyrotropin-Releasing Hormone; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium