Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the nonreceptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP␣. PTP␣ becomes activated in vivo after TPA stimulation. Further, we show that PKC␦ phosphorylates and activates only PTP␣ in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC␦ have a markedly reduced PTP␣ and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP␣ by PKC␦, which in turn dephosphorylates and activates Src.Protein kinase C (PKC) 1 is a family of phospholipid-dependent serine/threonine kinases comprising 10 isozymes differing in their molecular domain organization of up to 4 variable and 3 constant regions and in their functions. These PKC isozymes are subdivided into three classes: (i)the "conventional" cPKCs, PKC-␣, -␤ (␤I and ␤II), and -␥, which can be activated by phosphatidylserine (PS), diacyl glycerol (DAG), or phorbol esters through binding to the C1 domain and Ca 2ϩ through binding to a Ca 2ϩ -binding site in their second constant region, C2; (ii) the "novel" nPKCs, PKC-␦, -⑀, -, -, which lack the C2 region and thus are Ca-independent but still DAG-, PS-, and phorbol ester-responsive; and (iii) the "atypical" aPKCs, PKC-/ and -, which also lack the C2 region and, in addition, are devoid of a functional DAG-binding site. Hence, the atypical PKCs are only responsive to PS but not to DAG or phorbol ester (reviewed in Refs.

Protein kinase C, a multigene family of phospholipiddependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase C␦, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase C␦ fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3-5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase C␦ also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase C␦-overexpressing cells. Stimulation of overexpressed protein kinase C⑀ did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.

The catalytic domain of overexpressed protein kinase C (PKC)-δ mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-δ/εV5 and -ε/δV5, by swapping the V5 domains of PKC-δ and -ε. PKC-δ/εV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-δ's functionality. The other chimera, PKC-ε/δV5, endowed inactive PKC-ε with nearly all PKC-δ's apoptotic ability, confirming the importance of PKC-δ in this function. Green fluorescent protein (GFP)-tagged PKC-δV5 and -ε/δV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-ε and -δ/εV5 showed significantly less, indicating that the V5 region of PKC-δ contains determinants critical to its nuclear distribution. PKC-ε/δV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-ε, implicating the PKC-εV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-δ and -ε.

No abstract