In order to map the molecular determinants that dictate the subcellular localization of human protein kinase C ␣ (hPKC␣), full-length and deletion mutants of hPKC␣ 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, hPKC␣-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKC␣ 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 hPKC␣-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 hPKC␣, 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 hPKC␣ 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 hPKC␣. Protein kinase C (PKC)1 is a term coined to designate a family of isoforms that play key roles in the processes of proliferation/apoptosis, differentiation, or hormone release and the function of which is regulated at multiple levels: transcription, phosphorylation, and subcellular targeting.Subcellular targeting of PKC, and particularly that of the conventional PKC ␣, ␤, and ␥ isoforms, is linked to enzyme activation. Indeed, inactive PKC is mostly cytoplasmic, whereas activated PKC translocates to various membrane compartments, such as the plasma membrane. Physiological activation of the conventional PKC is associated with an increase in diacylglycerol (DAG) and intracellular Ca 2ϩ concentrations (1), which results from the activation of a seven transmembrane receptor coupled to phospholipase C␥ via a heterotrimeric G protein (2). The increase in Ca 2ϩ is thought to be essential for translocation, although it can be bypassed by the phorbol ester phorbol 12-myristate 13-acetate (PMA) (3). The increase in DAG concentration at the plasma membrane allows additional conformational changes to achieve PKC activation at its targeting site. When inactive, PKC is in a "closed" conformation due to the interaction of the pseudosubstrate sequence with the catalytic site (4, 5). ...

In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation.

Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase C␣ (PKC␣) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKC␣ (hPKC␣) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKC␣, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca 2؉ ] i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKC␣ wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and ␤-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKC␣ translocates but did not accumulate at the plasma membrane and ␤-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKC␣ at the cell-cell contacts is a regulated process which depends upon the integrity of both PKC␣ and the actin microfilament network.Several years ago, we have shown that in a cell subpopulation of human pituitary and thyroid tumors, protein kinase C␣ (PKC␣) bore a point mutation at position 294, resulting in the substitution of an aspartic acid by a glycin (2, 31). The analysis of the biochemical properties of the D294G mutant and of the phenotype of embryonic fibroblasts stably transfected with it revealed a selective loss of recognition of substrates having characteristics of anchoring proteins (32) and a dramatic decrease in the dependence on serum growth factors for proliferation (3). In Rat6 fibroblasts stably transfected with human PKC␣ (hPKC␣) or its mutant and treated with phorbol 12-myristate 13-acetate (PMA) for 1 h, the D294G mutant localized in the lysosome compartment (unpublished data), whereas wild-type hPKC␣ (hPKC␣-wt) localized at the plasma membrane but not selectively at cell-cell contacts (3). Fibroblasts and epithelial cells are very different in many features. We therefore changed our model to the GH3B6 epithelial pituitary cell line. In this cell line, we found that PKC␣ is selectively targeted to the cell-cell contacts upon thyrotropinreleasing hormone (TRH) or PMA stimulation (42). To our knowledge, there is only one other study reporting on the presence of PKC␣ at the cell-cell contacts during spontaneous or...

Protein kinase C (PKC) has been implicated in the control of intercellular adhesion. Our previous observation demonstrating that activated PKC alpha (PKCα is selectively targeted to cell-cell contacts of pituitary GH3B6 cells supports these findings. The relevance of this observation is further strengthened by the present data establishing that this targeting selectivity also occurs in the pituitary gland. Moreover, a new mechanism involved in the control of PKC targeting is unravelled. We demonstrate that a three amino acid motif located in the V3 region of α and epsilon (ϵ (GDE/GEE respectively) is essential for the targeting selectivity of these isoforms because: (1) this motif is absent in delta (δ) and mutated in the natural D294GPKCα mutant, which do not exhibit such selectivity, and (2) a GEE to GGE mutation abolishes the selectivity of targeting to cell-cell contacts for ϵ, as it does for the D294G PKCα mutant. Thus the GD(E)E motif may be part of a consensus sequence able to interact with shuttle and/or anchoring proteins. GFP-tagged deletion mutants also reveal a new function for the pseudosubstrate in the cytoplasmic sequestration. Together, these data underline the complexity of PKC subcellular targeting in the pituitary, determined by the cell-cell contact, at least for α and ϵ