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Inhibition by Sulfatide of 21-Kda Protein Phosphorylation by Protein Kinase C in Cow Mammary Gland and Its Reversal by Phosphatidylserine

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Inhibition by Sulfatide of 21-Kda Protein Phosphorylation by Protein Kinase C in Cow Mammary Gland and Its Reversal by Phosphatidylserine FULL PAPER Biochemistry Inhibition by Sulfatide of 21-kDa Protein Phosphorylation by Protein Kinase C in Cow Mammary Gland and its Reversal by Phosphatidylserine Norio KATOH1) 1)National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan (Received 3 December 2003/Accepted 24 February 2004) ABSTRACT. The effect of sulfatide, a sulfated sphingolipid, on phosphorylation of endogenous proteins by protein kinase C (PKC) was examined in cow mammary gland. Several proteins, including 21-kDa, 43-kDa and 56-kDa proteins in the cytosolic fraction, were f ound to be substrates for PKC by phosphorylation in the absence or presence of the cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG), phosphati- dylserine (PS) and Ca2+. Sulfatide inhibited the 21-kDa phosphorylation, whereas it enhanced the 56-kDa and 43-kDa phosphorylation. Experiments were then conducted to examine whether other sphingolipids, including sphingosine, dihydrosphingosine, ceramides, galac- tocerebrosides, psychosine and sphingomyelin, modulated phosphorylation of the PKC substrates. Sphingosine, dihydrosphingosine and psychosine did not inhibit the 21-kDa phosphorylation; however, they enhanced the 56-kDa and 43-kDa phosphorylation. Ceramides, galactocerebrosides and sphingomyelin did not inhibit the 21-kDa or enhance the 56-kDa and 43-kDa phosphorylation. The inhibition by sulfatide of the 21-kDa phosphorylation was reversed by excess addition of PS, but not by OAG or Ca2+; whereas the enhancement by sulfatide, as well as sphingosine, dihydrosphingosine and psychosine, of 56-kDa and 43-kDa phosphorylation was not affected by PS, OAG or Ca2+. It is suggested that sulfatide is involved in the regulation of PKC-dependent phosphorylation by modulating the associ- ation of PKC substrates, in particular the 21-kDa protein, with membrane phospholipids in cow mammary gland. KEY WORDS: cattle, mammary gland, protein kinase C, sphingolipids, sulfatide. J. Vet. Med. Sci. 66(7): 821–825, 2004 Sphingolipids, a major class of membrane lipids, consist phorylation in cow mammary gland. of a hydrophobic portion, called ceramide, which contains a mixture of fatty acids that are amide-linked to sphingosine MATERIALS AND METHODS or other related long-chain aliphatic amines (sphingoid bases), and a hydrophilic portion (polar head group) [30]. Materials: Sulfatide, sphingosine, dihydrosphingosine, Except for a few cases such as phosphorylcholine in sphin- ceramides, galactocerebrosides, psychosine, sphingomye- gomyelin, most of the polar head groups are carbohydrates, lin, 1-oleoyl-2-acetyl-sn-glycerol (OAG), PS, lysine-rich and glycosphingolipids include neutral lipids (galactocere- histone (type III-S), phenylmethylsulfonyl fluoride (PMSF) brosides), and acidic lipids, which contain sialic acid resi- and leupeptin were purchased from Sigma Chemical Co. dues (gangliosides), or sulfate monoester groups (St. Louis, MO, U.S.A.). [γ-32P]ATP was from New (sulfatides). Sulfatide is well known to be the causal factor England Nuclear-DuPont (Wilmington, DE, U.S.A.). Cow for metachromatic leukodystrophy in the central and periph- mammary glands in the midlactating stage (150 to 200 days eral nervous systems [31]. after parturition) were obtained from a local slaughterhouse. Protein kinase C (PKC) is an enzyme activated by dia- The pars glandularis was cut into small blocks, and quickly cylglycerols, phospholipids, in particular phosphati- frozen at –80°C until use. dylserine (PS), and Ca2+, and exerts a wide variety of Phosphorylation: Mammary gland tissue (1 g) was cellular functions by phosphorylating serine and threonine thawed, cut into small pieces and homogenized in 9 volumes residues of various enzymes and proteins [13, 20, 23]. Sph- of 0.25 M sucrose containing 20 mM Tris-HCl (pH 7.5), 2.5 ingolipids are modulators for PKC [5]. Sphingosine inhibits mM ethyleneglycol bis(2-aminoethylether)tetraacetic acid PKC activity when lysine-rich histone (histone H1), one of (EGTA), 10 mM 2-mercaptoethanol, 1 mM PMSF and 10 the most useful exogenous substrates, is used as the sub- µg/ml leupeptin, using a Polytron PTA 10S homogenizer strate [6]; however, in the case of endogenous protein phos- [8]. The homogenate was centrifuged at 105,000 × g for 60 phorylation, sphingosine acts as inhibitors or stimulators for min; the resultant supernatant referred to as cytosol. The individual substrates [9, 26]. Moreover, gangliosides have pellet was treated with 0.3% Triton X-100 for 60 min, then dual effects on phosphorylation of various PKC substrates centrifuged at 105,000 × g for 60 min, and the supernatant in rat brain [3] and cow mammary gland [11, 12, 15, 16]. obtained was used as the (solubilized) total particulate frac- Sulfatide, on the other hand, appears to inhibit PKC activity tion. when the histone is included in the assay mixture [32]. In Phosphorylation of endogenous substrate proteins was regard to endogenous substrates, effects of sulfatide on pro- carried out as described previously [9]. Briefly, the standard tein phosphorylation have not yet been determined. The reaction mixture (0.2 ml) contained 25 mM Tris-HCl (pH purpose of the present study was to examine whether sul- 7.5), 10 mM MgCl2, 0.25 mM EGTA, 10 mM 2-mercapto- fatide modulates PKC-dependent endogenous protein phos- ethanol, 50 µM [γ-32P]ATP, 20 µl of cytosolic (containing 822 N. KATOH 110–180 µg of proteins) or particulate fraction (30–50 µg of Table 1. Effect of sulfatide on PKC activity µ µ µ protein), with or without 1 g of OAG, 5 g of PS and 1 M µ 2+ Sulfatide ( g/ml) PKC activity (pmol/min) Ca . Sulfatide and related sphingolipids suspended in 20 –CF +CF mM Tris-HCl (pH 7.5) were sonicated and added to the mix- ture. The reaction was initiated by the addition of ATP and 02.6511.7 continued for 5 min at 30°C. Phosphorylated proteins were 12.1611.9 25 2.57 11.8 separated by sodium dodecyl sulfate-polyacrylamide gel 50 2.74 11.3 electrophoresis (SDS-PAGE), using a 12% constant gel, 100 2.82 10.1 then visualized by autoradiography. Cytosolic PKC was partially purified by DEAE-cellulose Other methods: PKC from mammary gland cytosol was chromatography, and the eluant (2 µg/0.2 ml) was used for the partially purified by DEAE-cellulose chromatography [10]. assay. In addition, lysine-rich histone (40 µl/0.2 ml) was included Calmodulin was purified from pig testis [19]. Protein was in the mixture. Reactions were initiated by the addition of ATP determined by the method of Bradford [1]. and proceeded for 5 min at 30°C, which was in the linear phase of the time course. All values are means of two separate RESULTS experiments and each assay was done in duplicate. CF, PKC cofactors (5 µg/ml OAG, 25 µg/ml PS and 1 µM Ca2+). When lysine-rich histone was used as the substrate, the effect of sulfatide on PKC activity was examined (Table 1). kDa proteins were also found to be PKC substrates (Fig. 2, Sulfatide had little effect on PKC activity within the range lane 1). Of the PKC substrates, the 36-kDa protein was of 1 to 100 µg/ml, suggesting that sulfatide did not directly identified as annexin I [14, 16, 17]. A 97-kDa protein was interact with the histone substrate. stimulated only by Ca2+ alone and seemed to be phosphory- Phosphorylation of cytosolic proteins by endogenous lase b (the substrate for calmodulin-sensitive phosphorylase protein kinases and subsequent autoradiography revealed b kinase [9]). Phosphorylation of 30-kDa casein (the sub- that 91-kDa, 72-kDa, 56-kDa, 43-kDa, 36-kDa, 27-kDa, 24- strate for casein kinases) was independent of the cofactors. kDa, and 21-kDa proteins were substrates for PKC because Phosphorylation of the 21-kDa protein was found to be of their enhanced phosphorylation by the cofactors (Fig. 1, inhibited by sulfatide (Fig. 1). Inhibition of the 24-kDa lanes 1 and 2). Although unclear in Fig. 1, 89-kDa and 41- phosphorylation was similarly observed. Phosphorylation Fig. 1. Effect of sulfatide on phosphorylation of endogenous proteins from the cytosolic fraction of cow mammary gland and its reversal by the PKC cofactors. CF, cofactors (5 µg/ml OAG, 25 µg/ml PS, and 1 µM Ca2+); OAG, (50 µg/ml OAG, 25 µg/ml PS, and 1 µM Ca2+); PS, (250 µg/ ml PS, 5 µg/ml OAG, and 1 µM Ca2+); Ca, (250 µM Ca2+, 5 µg/ml OAG, and 25 µg/ml PS); S, sulfatide. Endogenous protein kinases were used as the enzyme source. The phosphorylation experiment was repeated three times. A representative autoradiogram is shown. INHIBITION BY SULFATIDE OF PKC PHOSPHORYLATION 823 of the 56-kDa and 43-kDa proteins, on the other hand, was gosine inhibited phosphorylation of the 97-kDa, 91-kDa and inversely enhanced by sulfatide. The other PKC substrates 89-kDa proteins but enhanced that of the 56-kDa, 43-kDa, (such as the 91-kDa and 27-kDa proteins) appeared to be 27-kDa, 24-kDa, and 19-kDa proteins. Dihydrosphingosine resistant to sulfatide. The inhibition by sulfatide of the 21- did not inhibit the 97-kDa, 91-kDa or 89-kDa phosphoryla- kDa phosphorylation by PKC was effectively reversed by tion but it enhanced that of the 56-kDa, 43-kDa, 27-kDa and the excess addition of PS (Fig. 1, lane 9), but not by OAG 24-kDa proteins. Psychosine inhibited the 97-kDa phospho- (lane 8) or Ca2+ (lane 10), suggesting that sulfatide inhibited rylation, while it, like dihydrosphingosine, enhanced that of the 21-kDa phosphorylation competitively with respect to the 56-kDa, 43-kDa, 27-kDa and 24-kDa proteins. Ceram- PS. Reversal by PS of the 24-kDa protein was obscure. ides III and IV, galactocerebrosides I and II, and sphingo- When calmodulin was included instead of PS, the inhibition myelin had little effect on the PKC substrates.
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