Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membrane‐bound receptors

Abstract

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC α along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC ɛ. In contrast, thrombin had a smaller effect on nuclear translocation of PKC α and did not influence PKC ɛ, but instead induced a rapid nuclear translocation of PKC ζ. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC α and ɛ within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms α, δ,ɛ and ζ in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC α and ɛ to the perinuclear region and into the nucleus, while PKC δ and ζ showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC α and ɛ was reduced to or below control values. At 8 h, increased nuclear expression of isoforms α,ɛ and ζ was observed, while isoform δ was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.