Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells

Miłoszewska, Joanna; Trawicki, W.; Janík, P.; Moraczewsk, Jerzy; Przybyszewska, Małgorzata; Szaniawska, B

doi:10.1016/0014-5793(86)80997-8

Cited by 20 publications

(8 citation statements)

References 6 publications

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“…Redistribution of the enzyme was very rapid, occurring within 5-10 min of stimulation. The rapid membrane association of PKC in quiescent EC treated with fetal calf serum is in agreement with the observations of Miloszewska et al (1986) who studied a fibroblastic line of cells. In the EC system, the membrane-associated PKC was maintained in this subcellular fraction for 2 h although a 15% loss of total PKC activity was observed.…”

Section: Discussionsupporting

confidence: 89%

Growth‐dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

Uratsuji

DiCorleto

1988

Journal Cellular Physiology

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We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.

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Section: Discussionsupporting

confidence: 89%

Growth‐dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

Uratsuji

DiCorleto

1988

Journal Cellular Physiology

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“…As expected, this treatment markedly densduction pathway, creased PKC enzyme levels ( Fig. 5A) (26,32) and signifiactive GTP-bound cantly reduced the basal level of OPN gene expression ( . Surprisingly, the concomitant ine nucleotide ratio induction of ras with PKC depletion restored basal OPN ted parental 10T½2 gene expression to the level of untreated samples (Fig.…”

supporting

confidence: 79%

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Krook¹,

Rapoport²,

Anderson³

et al. 1993

Mol. Cell. Biol.

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Both p21' and protein kinase C (PKC) are believed to function downstream of plasma membraneassociated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p2l' and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.Mammalian ras proteins (p2lras and Ras) play an important role in the control of cell proliferation and differentiation (38). However, their physiological role in signal transduction pathways that extend from ligand-receptor interactions at the cell surface to activation of gene expression in the nucleus is still incompletely understood. The observation that serum growth factor receptors as well as receptor and non-receptor-type oncogene products with tyrosine kinase (TK) activity require functional Ras to elicit mitogenic and oncogenic responses (2,8,27,29,46) suggests that p2lras acts downstream of TK. Protein kinase C (PKC), a ubiquitous phospholipid and Ca2+-dependent serine-threonine kinase (31), also functions downstream of TK along the phospholipase C-mediated pathway of phosphatidylinositol lipid (PI) turnover (14)(15)(16)30 parts (34,53). Scrape-loading experiments have demonstrated that a very early effect of p2lras is to activate PKC (28). Moreover, microinjection of antibodies against phospholipase C, an upstream effector of PKC activity in the PI pathway, inhibits DNA synthesis induced by Ras (47). These observations support the model of a linear signal transduction pathway in which p2lras activation precedes PKC function. However, the following experiments have prompted the opposite conclusion. Although transforming Ras alone does not stimulate a mitogenic response in cells in which PKC has been downregulated (20), the Ras-neutralizing monoclonal antibody (MAb) Y13-259 blocks TPA-induced mitogenesis in quiescent fibroblasts (55). Moreover, in T lymphocytes the activation of PKC enhances the activational state of p2lras, presumably by relaxing the effect of the regulatory GTPase-activating protein Ras.GAP (6). The latter data suggest that p2lras is positioned downstream of PKC in a linear signaling pathway.Most of the studies described above evaluated Ras and PKC function by analysis of DNA synth...

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“…Augmented basal 3H-thymidine incorporation by neonatal PA SMC led to significant increases in cell number (s2.5-fold). Auto-activation of PKC may have contributed to the persistent DNA synthesis (Miloszewska et al, 1986). This property of continued growth in response to a previous stimulus or injury could have significant implications in vivo.…”

Section: Discussionmentioning

confidence: 99%

Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: Dependence on cell size, time from birth, insulin‐like growth factor I, and auto‐activation of protein Kinase C

Dempsey

Badesch

Dobyns

et al. 1994

Journal Cellular Physiology

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Based on the unique susceptibility of the neonatal pulmonary circulation to hypoxia-induced structural alteration in vivo, we hypothesized that pulmonary artery (PA) smooth muscle cells (SMC) from the neonate would demonstrate enhanced growth capacity in vitro compared to adult cells. To test this hypothesis, matched neonatal and adult bovine SMC were tested for differences in size, serum-stimulated proliferation, susceptibility to senescence, resistance to serum withdrawal, autocrine growth capacity, and responsiveness to a locally important growth factor (insulin-like growth factor I; IGF-I) and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA). Neonatal PA SMC were smaller, grew faster, reached a higher plateau density, and were less susceptible to senescence. They were more resistant to serum withdrawal, had spontaneous autocrine growth capacity, and were more responsive to IGF-I, PMA, and the combination. Acquisition of increased growth factor responsiveness occurred between d5 and d14 after birth. Increased neonatal growth to IGF-I was associated with reduced IGF-I binding activity, implicating a post-receptor mechanism in enhanced responsiveness. Increased membrane-bound PKC catalytic activity was found in serum-deprived neonatal SMC. This basal increase was equal to that stimulated by 1 nM PMA in adult SMC, a pretreatment that caused these cells to become as responsive to IGF-I as untreated neonatal ones. We conclude that neonatal bovine PA SMC have marked enhancement of growth capacity in vitro, the acquisition of which is dependent on time from birth and is associated with auto-activation of PKC, These increased growth properties detected in vitro may contribute to the striking hyperplasia of neonatal PA SMC found in vivo following hypoxic exposure.

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Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells

Cited by 20 publications

References 6 publications

Growth‐dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

Growth‐dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: Dependence on cell size, time from birth, insulin‐like growth factor I, and auto‐activation of protein Kinase C

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