Fitzer, C.J., O'Brien, A.O., Guillem, J.G., Weinstein, LB. (1987) ... Guillem, J.G., O'Brian, C.A., Fitzer, J., Jonhson, M.D., Forde, K.A., LoGerfo, P. and. Weinstein ...
Vol. 41, No. 2, February 1997
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages 329-337
URSODEOXYCHOLATE PROMOTES PROTEIN PHOSPHORYLATION IN THE CYTOSOL OF RAT HEPATOCYTES 1. Ferdinando Capuano 2, Michele Barone*, Nicola D'Eri, Elisabetta Russo, Davide Varone, Antonio Francavilta* and Sergio Papa Institute of Medical Biochemistry and Chemistry Gastroenterology, University of Bari, Bari, Italy
and
*Department
of
Received November29, 1996
Summary A study is presented of the effect of the bile salt ursodeoxycholate (UDC) on protein phosphorylation by [~,-32P]ATP in the cytosol from rat hepatocytes. Gel electrophoresis and corresponding autoradiograms of cytosolic proteins show that UDC promotes phosphorylation of at least eight different protein bands. Four of them (the 36, 60, 64 and 76 kDa) are phosphorylated by Ca 2+ and phospholipid-dependent protein kinase (PKC); three (the 31, 51 and 71 kDa) are phosphorylated by cAMPdependent protein kinase (PKA) and one protein band, with molecular weight of 34 kDa, apparently contains substrates of both PKC and PKA. Data are reported indicating that UDC can directly affect the intrinsic activity of protein kinases. Key words Bile salt, rat hepatocytes, protein phosphorylation, cAMP-dependent protein kinase, Ca 2+ and phospholipid-dependent protein kinase
Introduction Reversible protein phosphorylation plays a control role in signal transduction pathways and regulation of cellular functions [1]. The eukaryotic protein kinases responsible for protein phosphorylation include a number of enzyme families different for regulatory systems, subunit structure, subcellular localization and substrate specificity [1-4].
1. Supported by grants of the Italian Research Council, Special Project A.C.R.O., Contract 104283. 39.9410582 Abbreviations: UDC, ursodeoxycholate;PKA, cAMP-dependentprotein kinase; PKC, Caz+ and phospholipid-dependent protein kinase; DAG, diacylglycerol;PL, phospholipid; PS, phosphatidylserine;SDSPAGE, sodiumdodecylsulfatepolyacrylamidegel electrophoresis. 2. To whom requests for reprints should be addressed, at Institute of Medical Biochemistryand Chemistry, Universityof Bari, PiazzaGiulioCesare, 70124 Bari, Italy 1039-9712/97/020329-09505.0010 329
Copyrighl 9 1997 tLVAcademic Press Au,r All ~qghts q/reproduction it* any form reserved
Vol. 41, No. 2, 1997
Once activated by external
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stimuli, protein kinases phosphorylate a large variety of
target proteins modulating their properties. This, in turn, results in cytosolic and nuclear events that control metabolic pathways, cellular differentiation and growth, gene expression and oncogenesis [5]. Several lines of evidence suggest that bile salts act as tumor promoters in colon cancer [6,7] by increasing cell proliferation probably through different mechanisms [811]. Enhanced proliferative activity of colonic mucosa by bile salts has been associated with alterations in the state of activation of the Ca 2+ phospholipid-dependent protein kinase C [11,12] suggesting the involvement of this cellular signalling system in the promoting effect of bile salts. Recently, it has been reported that bile salts stimulate proliferation in cultured hepatocytes. In particular, ursodeoxycholate (UDC) at physiological concentrations, stimulated proliferation of rat hepatocytes cultured either in basal conditions or in the presence of mitogens [13]. Based on these results, further confirmed in vivo [14], we attempted to define the mechanism mediating this stimulatory effect. This paper reports a
study of the effect of ursodeoxycholate on protein
phosphorylation in the cytosol from rat hepatocytes. Results are presented showing that ursodeoxycholate stimulates the ]2p-incorporation into several proteins via both cAMP- and Ca 2+ and phospholipid-dependent phosphorylation.
Materials and Methods Chemicals. [y-32p]ATP (3,000 Ci/mmol) and Hyperfilm-MP were from Amersham International; collagenase type I was from Worthington Diagnostic System, Freehold, NJ; diacylglycerol, phosphatidylserine, cAMP and the catalytic subunit of cAMP-dependent protein kinase purified from bovine heart were from Sigma; histone H2B was from Boehringer-Mannheim; ursodeoxycholate was from Calbiochem. All other reagents were of the highest purity grade commercially available. Animals. Male (F-344) rats, weighing 150-200 g, were obtained from Charles River, Calco (CO), Italy. They were kept in a temperature and light-controlled room (light on from 7 a.m. to 7 p. m.) for at least one week before being used and received food and water ad libitum.
Hepatocyte isolation. Hepatocytes were isolated by modification of the in situ two-step collagenase perfusion technique of Seglen [15], as previously described [16]. The purity of the preparation was more than 98%, as demonstrated by the staining for glucose-6-phosphat ase [16]. 32p-labelling of cytosolic protein extract. Freshly isolated hepatocytes were homogenized, at 4 ~ in a buffer containing: 250 mM sucrose, 1 mM EDTA, 1 mM Tris-HC1 at pH 7.5. Hepatocyte homogenates were subjected to differential centrifugation and the 100000 x g supernatants used for the assay. Cytosolic protein
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phosphorylation was assayed essentially as described in [17]. A fixed amount of protein (300 pg) was incubated, under stirring, for 20 rain at 37 ~ in 300 btl of a basic mixture containing: 20 mM Tris-HC1, pH 7.5, 8 mM MgC12, 20 mM NaF, 1.5 pg oligomycin and 70 btM [y-32p]ATP (500 cpm/pmol), cAMP (50 pM) and EGTA (0,5 raM) were added to the incubation mixture to evaluate cAMP-dependent phosphorylation. 0.125 mM CaC12, 0.8 pg/ml diacylglycerol and 44.4 pg/ml phosphatidylserin, instead of cAMP and EGTA, were added to evaluate Ca 2+ and phospholipid-dependent phosphorylation. To stop the reaction, 150 pl of the suspension was mixed with 40 j.tl of 50 mM Tris-HC1 pH 6.8, 10% v/v glycerol, 1% w/v SDS, 2% 13-mercaptoethanol and boiled for 3 min. Gel electrophoresis and autoradiography. Boiled samples were subjected to gel electrophoresis on slabs of 9% polyacrylamide plus 0.1% SDS, essentially as described in [18]. Gels were fixed in 50% methanol plus 10% acetic acid, stained with 0.1% Comassie blue, destained in the same mixture, photographed and dried for autoradiography. For molecular weight assignment, Bio-Rad standards were used. Radioactive bands were visualized by exposure to Hyperfilm (Amersham). Autoradiographs were scanned at 590 nm with a Camag Densitometer.
Results Fig. 1 shows the 32p-labelling patterns (A), and the corresponding densitograms (B), of PAGE-resolved protein bands from control and UDC- treated cytosolic extracts, freshly prepared from rat hepatocytes. Incubation of the cytosol with [y-32p]ATP (Fig.lA, lane 1) resulted in faint protein labelling , except for the protein band with apparent molecular weight of 60 kDa (detected by Comassie blue, not shown). The presence of UDC in the incubation medium (Fig. 1A, lane 2), did not greatly alter the phosphorylation pattern of cytosolic proteins except the 60 kDa protein band, whose phosphorylation in some experiments was depressed. cAMP (plus EGTA) added to the cytosolic extract during incubation promoted the 32p-labelling of various protein bands in a wide range of molecular weights (Fig. IA, lane 3). In particular, cAMP gave considerable phosphorylation of the protein bands with apparent molecular weights of 64, 60, 57 and 51 kDa. The presence of UDC in the incubation mixture further increased the phosphorylation of the protein band of 51 kDa (Fig. 1A, lane 4; see also Fig. IB). Furthermore, UDC greatly promoted, in the presence of cAMP, 32p-labelling of the bands with molecular weights of 71, 34 and 31 kDa. Phosphorylation of a group of protein bands, including the 57, 48, 46 and 43 kDa, was stimulated in the presence of cAMP in response to UDC, but not constantly and to a lower extent. Incubation of the cytosolic protein extract with [2-32p]ATP in the presence of Ca 2+ (plus DAG and PL), gave labelling of several protein bands in the range of molecular weights of 100 to 36 kDa as shown by autoradiograms (lane 3) and
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