A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

Abstract

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...

A Spatiotemporally Coordinated Cascade of Protein Kinase C Activation Controls Isoform-Selective Translocation

“…3B and C): 118 s for PKC␤1, 20 s for PKC␣, 15 to 20 min for PKCε, and 33 s for PKC␦ (see the videos in the supplemental material). Strikingly, the time during which PKC␤1 remained activated was identical to that of the D294G PKC␣ mutant (51). The fact that PKC␣ mutant activation is phase locked with [Ca 2ϩ ] i variations (51) suggests that the PKC␣ mutant has indeed switched from a Ca 2ϩ -independent to a Ca 2ϩ -dependent translocation as hypothesized above.…”

“…Strikingly, the time during which PKC␤1 remained activated was identical to that of the D294G PKC␣ mutant (51). The fact that PKC␣ mutant activation is phase locked with [Ca 2ϩ ] i variations (51) suggests that the PKC␣ mutant has indeed switched from a Ca 2ϩ -independent to a Ca 2ϩ -dependent translocation as hypothesized above. Conversely, the D187/246/248N PKC␤1 mutant, which accumu- The selective accumulation of PKC␣ and -at cell-cell contacts is dependent on PKC activity.…”

“…In GH3B6 cells, the fact that PKC␣ relocated to the cytoplasm before the [Ca 2ϩ ] i decreased to baseline levels (52) also indicates that cPKC activation is not always phase locked with variations in calcium concentration as suggested in many studies, such as that of Violin et al, who also showed the coincidence of PKC substrate phosphorylation with calcium oscillations (54). In addition, we have shown that the selective targeting of PKC␣ to cell-cell contacts is probably finely regulated at the molecular level, since the natural D294G point mutation (located in the V3 region of the enzyme and thus not involved in calcium or DAG binding), previously identified in human pituitary and thyroid tumors (2,40,41), abolished the selective accumulation of PKC␣ at the cell-cell contact (51). The same result was obtained when this mutation was introduced in the PKCε coding sequence (43).…”

“…In order to answer this question, we analyzed the involvement of PKC␣ and -ε on the TRH-induced ␤-catenin relocation at cell-cell contacts previously reported for this cell line (51). Figure 10A shows the time course of ␤-catenin relocation at cell-cell contacts upon TRH stimulation.…”

Protein kinase Cα participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells

“…Moreover, it was reported that calponin, a cytoskeletal protein, may serve to regulate PKC by facilitating its phosphorylation (24). A recent study revealed that a functional and integral actin microfilament network is essential for translocation of PKC␣ from the cytosol to the plasma membrane (47).…”