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

Abstract

In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCβ1, PKCα, PKCε, and PKCδ, resulting in their accumulation at the entire plasma membrane (PKCβ and -δ) or selectively at the cell-cell contacts (PKCα and -ε). The duration of activation ranged from 20 s for PKCα to 20 min for PKCε. PKCα and -ε selective localization was lost in the presence of Gö6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCα localization is controlled by PKCβ1 activity and is calcium independent, while PKCε localization is dependent on PKCα activity. PKCδ was independent of the cascade linking PKCβ1, -α, and -ε. Furthermore, PKCα, but not PKCε, is involved in the TRH-induced β-catenin relocation at cell-cell contacts, suggesting that PKCε is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCβ1 activation, which in turn initiates a calcium-independent but PKCβ1 activity-dependent sequential translocation of PKCα and -ε. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Philippe Jay and Catherine Legraverend (IGF, Montpellier, France) and Daniel Fisher (IGMM, Montpellier, France) for their helpful criticisms.

This work was supported by the Ministère de la Recherche et de la Technologie, by the Association pour la Recherche contre le Cancer (ARC) (grant no. 5695). Barthélémy Diouf was supported by a grant from the ARC. Marion Peter was a recipient of a Long-Term Fellowship from the International Human Frontier Science Program Organization.