Protein kinase C (PKC) has been implicated in b1 integrin-mediated cell migration. Expression of the novel PKC isoform, PKCe, in PKCe ±/± cells is shown here to stimulate directional migration of cells towards b1 integrin substrates in a manner dependent on PKC catalytic activity. On PKC inhibition, integrin b1 and PKCe become reversibly trapped in a tetraspanin (CD81)-positive intracellular compartment, correlating with reduced haptotaxis. Immuno¯uorescence and pulse labelling studies indicate that this is a previously uncharacterized recycling compartment trapped by inhibition of PKC. Electron microscopy demonstrated the co-localization of PKCe and integrin b1 on the vesicular membranes. Finally, using a reconstituted in vitro system, the dissociation of PKCe from these vesicles is shown to be dependent on both the presence of cytosolic components and energy, and on PKC catalytic activity. The evidence presented indicates that PKCe controls an internal traf®c step that under uninhibited conditions permits the recycling of b1 integrin, contributing to cell motility.

Protein kinase C signaling is desensitized through a combination of dephosphorylation and proteolysis in intact cells. The process of dephosphorylation is analyzed here, as well as its relationship to degradation. It is established for protein kinase C␣ that dephosphorylation occurs in a membrane compartment following activation and temporally preceding significant degradation. The phosphatase responsible for the dephosphorylation appears to be a heterotrimeric type 2A phosphatase, which is shown to be in part constitutively membrane associated. Consistent with a role for this activity, okadaic acid is shown to inhibit the phorbol ester-induced dephosphorylation of protein kinase C that occurs in intact cells. Furthermore, phorbol esterinduced down-regulation of protein kinase C␣ is shown not to be dependent on the rate of dephosphorylation, indicating that these desensitizing pathways may operate in parallel.

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PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.