Cyclic amp‐dependent protein phosphorylation on the surface of rat hepatocytes

Sommarin, Marianne; Henriksson, Thomas; Jergil, Bengt

doi:10.1016/0014-5793(81)80225-6

Cited by 15 publications

(6 citation statements)

References 20 publications

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“…Data pointing to the existence of surface-located protein kinase have been obtained from a variety of cells (6)(7)(8)(9)26). The only indication published to our knowledge that phosvitin may release protein kinase from cells was interpreted as a result of cell leakiness (27), which was excluded in our studies.…”

Section: Discussionmentioning

confidence: 99%

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Substrate-effected release of surface-located protein kinase from intact cells.

Kübler¹,

Pyerin²,

Burow³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow without any morphological alteration. As (Heidelberg, Federal Republic of Germany), trypsin ("1:250") was from Difco. Heatand acid-stable inhibitor protein specific for catalytic subunits of cAMP-dependent protein kinases was isolated from rat skeletal muscle as described (13). 32P-Labeled phosvitin was obtained by incubation of phosvitin (5 mg/ml) and [ y-32P]ATP (0.5 ,uM; specific activity, 50 mCi/mmol; 1 Ci = 37 GBq) with intact HeLa cells for 15 min at 37°C. Cell supernatant was harvested and centrifuged at 2,500 x g for 10 min. Unbound [y-32P]ATP was removed from [32P]phosvitin by gel filtration (1.5 X 5 cm column; Ultrogel AcA 202 from LKB). Gel electrophoresis and autoradiography showed that phosvitin from different batches may possess either one or two major substrate components as evident from Figs. 1 and 3.HeLa cells were propagated in minimal essential medium/ 10% calf serum (Flow Laboratories) as described (5); embryo lung fibroblasts (HEL; human) were grown in basal medium Eagle's/10% fetal calf serum (Boehringer Mannheim). Hepatocytes from rat (provided by D. Mayer) were cultured in Ham's F12 medium/10% fetal calf serum; primary cultures from mouse cerebellum (provided by M. Schachner, Institute for Neurobiology, University of Heidelberg) were raised in basal medium Eagle's/10% horse serum (GIBCO Biocult). For experiments, cells were plated in plastic Petri dishes (Falcon; 3-, 5-, or 10-cm diameter). Synchronized HeLa cultures were obtained as described (14). Cell cycle traverse was monitored by flow cytometry (carried out by M. Stohr) as described (5). Cells were propagated free from mycoplasms.Preparation of cell supernatants was done at 37°C if not otherwise indicated. Cells were washed twice with prewarmed assay mixture (5 ml per 5-cm plate) consisting of 70 mM NaCl/ 30 mM TrisHOAc/5 mM Mg(OAc)2/5 mM potassium phosphate/0.5 mM EDTA/75 mM glucose, pH 7.2 (osmolarity, 290 ± 10 mOsm). Incubation was with 2 ml of assay mixture in the absence or presence of exogenous protein as given for the particular experiment. Cell incubation was done by moving the mixture gently over the cells (rocker platform; Bellco Glass) for various periods of time. Cell supernatants were centrifuged at 2,500 X g for 10 min and the flocculant was discarded. The remaining supernatant was kept in an ice bath until use. Control experiments in the absence of cells were carried out.For phosphorylation of cell surface protein in the absence or presence of exogenous protein substrate, intact monolayer cells were treated as described for the preparation of cell supernatants but incubation was in the presence of 0.5 ,uM [y_32p]_ ...

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Section: Discussionmentioning

confidence: 99%

“…A number of indications for the existence of surface-located protein kinase activity in other cell types have also been published (6)(7)(8)(9).…”

mentioning

confidence: 99%

Substrate-effected release of surface-located protein kinase from intact cells.

Kübler¹,

Pyerin²,

Burow³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Hepatocytes have PKAs on their surfaces and can secrete cyclic AMP (Barber and Butcher, 1983;Crooke et al, 1980;Kleine et al, 1987;Sommarin et al, 1981). Kleine et al (1987) reported that T51B rat liver cells accumulated the cyclic AMP-binding R subunits of PKAs on their surfaces during the G, build-up and then lost these R subunits before the onset of DNA replication, presumably because they bound cyclic AMP and then dissociated from the catalytic subunits.…”

Section: Discuss I 0 Nmentioning

confidence: 99%

“…First, hepatocytes pump cyclic AMP into their medium (Barber and Butcher, 1983). Second, cells such as hepatocytes and rat thymocytes have PKAs on their outer surfaces to receive secreted cyclic AMP (Kleine et al, 1987;Mac-Manus and Whitfield, 1972;Sommarin et al, 1981). Third, T51B rat liver cells load their surfaces with PKAs during the G, phase of their cycle and then lose the PKAs' cyclic AMP-binding regulatory (R) subunits from their surfaces near the end of the GI phase when they generate their GI cyclic AMP transient, but they do not lose the surface R subunits or start replicating DNA if the medium is Ca2+-deficient (Kleine et al, 1987).…”

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confidence: 99%

Extracellular calcium modulates prereplicative cyclic AMP surges in EGF‐stimulated primary neonatal rat Hepatocytes

Armato

Ribecco

Guerriero

et al. 1994

Journal Cellular Physiology

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The cells in nearly pure (96-98%) primary cultures of hepatocytes from neonatal rat liver in high (1.0 mM)-Ca2+, serum-free, synthetic HiWo5Ba2000 medium initiated DNA synthesis and entered mitosis between 11 and 30 h after the addition of 10 ng/ml EGF. During the 10-h prereplicative period, the cultured hepatocytes, like regenerating rat liver cells, generated two large cyclic AMP transients, one peaking between 30 min and 2 h and the other around 6 h. Hepatocytes stimulated by the same concentration of EGF in low (0.02 mM)-Ca2+ medium increased cyclic AMP synthesis as much as the EGF-treated hepatocytes in high-Ca2+ medium, but they released the additional cyclic AMP into the medium and could not generate prereplicative internal cyclic AMP surges, initiate DNA replication, or enter mitosis. These results suggest that one of the ways external Ca2+ controls prereplicative development of hepatocytes is to restrain the release of cyclic AMP and thus enable the cell to accumulate enough internal cyclic AMP to stimulate events required to initiate DNA replication.

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“…Several enzymes, such as ATPases, 5'-nucleotidases, proteases, and protein kinases, are located in or on the plasma membrane (Chiang et al, 1982;Kang et al, 1978Kang et al, , 1979Kinzel and Mueller, 1973;Kubler et al, 1982;MatManus and Whitfield, 1981;McPherson and Ramachandran, 1980;Mastro, 1979;Mastro and Rozengurt, 1976;Schlaeger and Kohler, 1976;Sommarin et al, 1981;Stanley et al, 1982;Wen et al, 1985). Of all these enzymes, the protein kinases seem to be especially important for proliferation, because several growth factors, such as EGF (Ushiro and Cohen, 1980), IGF-1 (Jacobs et al, 1983;Rubin et al, 1983), insulin (Kasuga et al, 1982), and PDGF (Ek et al, 1982;Nishimura et al, 19821, as well as the Avian sarcoma virus's mitogenid oncogenic pp6OwSrc product (Collett and Erikson, 1978;Whitfield, 1984, 1985), stimulate the phosphorylation of plasma membrane proteins, and a burst of membrane protein phosphorylation precedes the onset of DNA replication in Swiss albino 3T3 mouse cells and regenerating rat liver cells Mastro and Rozengurt, 1976).…”

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confidence: 99%

Ca²⁺‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis

Kleine

Whitfield

Boynton

1986

Journal Cellular Physiology

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Incubating T51B rat liver cells in Ca2+-deficient, serum-rich medium containing only 0.02 mM Ca2+ strikingly decreased the phosphorylation of several trypsin-removable cell surface proteins and arrested the cells in late G1 phase. Raising the Ca2+ concentration in the Ca2+-deficient medium from 0.02 mM to 0.5 mM or adding 80 nM TPA (12-O-tetradecanoyl-phorbol-13-acetate), a protein kinase C activator, stimulated the phosphorylation of a certain set of surface proteins within 5 min and the initiation of DNA replication within the next 2 hr. By contrast, incubation in the same Ca2+-deficient medium, which does not affect the proliferation of neoplastic T51B-261B cells, did not reduce the phosphorylation of cell surface proteins. These observations suggest that the stimulation of a Ca2+-dependent protein kinase (possibly protein kinase C) directly or indirectly phosphorylates certain cell surface proteins that might be part of the mechanism that triggers the Ca2+-dependent G1----S transition of normal cells. They also suggest that an alteration of this Ca2+-dependent protein kinase might be the reason for neoplastic cells being able to proliferate in the face of an external Ca2+ shortage that would stop the proliferation of normal cells.

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Cyclic amp‐dependent protein phosphorylation on the surface of rat hepatocytes

Cited by 15 publications

References 20 publications

Substrate-effected release of surface-located protein kinase from intact cells.

Substrate-effected release of surface-located protein kinase from intact cells.

Extracellular calcium modulates prereplicative cyclic AMP surges in EGF‐stimulated primary neonatal rat Hepatocytes

Ca²⁺‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis

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Cyclic amp‐dependent protein phosphorylation on the surface of rat hepatocytes

Cited by 15 publications

References 20 publications

Substrate-effected release of surface-located protein kinase from intact cells.

Substrate-effected release of surface-located protein kinase from intact cells.

Extracellular calcium modulates prereplicative cyclic AMP surges in EGF‐stimulated primary neonatal rat Hepatocytes

Ca2+‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis

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Ca²⁺‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis