190s Biochemical SocietyTransactions ( 1 992) 20
Isolation and biochemical characterization of protein kinase C from rat mammary gland S.LAVANDERO. E.MONCADA and M. SAPAG-HAGAR Departmento de Bioqufmica y Biologfa Molecular, Facultad de Ciencias Qufmicas y Famduticas. Universidad de Chile, Casilla 233, Santiago, Chile.
Protein phosphorylation is a prominent mechanism by which hormones and growth factors modulate cellular functions[1,2]. One of the major transduction pathways, receptor-mediated hydrolysis of phospholipids, leads to the activation of protein kinase C (PKC)[3,4]. PKC is widely distributed and its activity is diacylglycerol-. phospholipid-. and calcium- dependent[5]. Accumulating evidence, both direct and indirect, indicates that PKC plays an imponant role in the development of mammary tissue[6-10]. Mouse mammary PKC activity shows developmentrelated regulation. a decrease being observed during pregnancy and throughout lactation[6]. Because of the role of PKC in mammary growth and differentiation, we have purified and characterized the enzyme from this tissue. Mammary PKC was purified from Sprague-Dawley rats in midpregnancy, the stage at which activity is maximal (Lavandero. S.. Moncada, E.,Foncea, R., and Sapag-Hagar, M.. unpublished work). by a three-step procedure essentially as described by Woodgea er al.[ll]. The PKC activity was measured by the incorporation of 32P from [32P]ATP into Hl-histone[l2]. Protein concentrations were determined by the Bradford method[l3]. Sodium dodecyl sulphatepolyacrylamide electrophoresis and isoelectric focusing were performed as described previously[l4.15). On DEAE column chromatography. PKC eluted as a peak in the first fractions at 20-100mM-NaO. On Phenyl Sepharose. the enzyme eluted as a sharp peak at an ammonium sulphate Affinity chromatography using concentration close to zero. phosphatidylserine-acrylamide yielded a sharp peak of activity. Table 1 summarizes the purification data. Silver staining of polyacrylamide gels containing sodium dodecyl sulphate showed a protein band with an apparent molecular weight of 78kDa; isolectric focusing gave two bands with isoelectric points of 5.8 and 5.9. Both values agree with those obtained for PKC from other sources[3J.16]. Because of the instability of this preparation, however, partiallypurified enzyme was used in the subsequent biochemical characterization. Mammary PKC activity was dependent on phosphatidylserine and Ca2+and showed a marked concentrationdependent increase in the presence of diolein or 12-0-tetradecanoylphorbol 13-acetate. Table 1.
One unit (U) of activity is defined as that amount of PKC that 27°C. Volume
Rotcin conc.
Total Activity
Specific Activity
Purification
Yield
(ml)
(mg/ml)
(V)
(U/mg)
(fold)
(%)
Extract
14
9.03
0.81
0.01
DEAE cellulose
45
1.12
12.83
0.25
I .o
100
Phenyl Sepharose
65
0.21
11.05
0.81
3.2
86
PS-anyla-
2
0.02
0.06
1.40
5.5
4
mi&
We are indebted to Dr. C.I. Fogson for his help in the preparation of this manuscript. 'Ihis work was supported by Grants Fondecyt 91-0879 and DTI Universidad de Chile B-3113-9013. S.L. is a recipient of a fellowship from Fundaci6n Andes. 1. Nestler, E.& Greengard. P. (1984) Science 225.1357-1364. 2. Hunter, T. &Cooper. J.A. (1985) Annu.Rev.Biochem. 54,897930. 3. Nishizuka, Y.(1988) Nature 334,661-665. 4. Benidge, M.J. (1987) Annu.Rev.Biochem. 56,159-193. 5. Nishizuka, Y.(1986) Science 233,305-312. 6. Caulfield, J.J. & Bolander, F.F. (1986) J.Endocrino1.109,29-34. 7. Katoh, N. (1990) J.Dairy Sci. 73,1201-1207. 8. Holladay. C.S. & Bolander, F.F. (1986) Pm.Soc.Exp.Biol.Mcd. 183,343-347. 9. Waters, S.B. & Rillema, J.A. (1989) Mol.Cell.Endocrinol. 63.159-166. 10. Taketani, Y.& Oka, T. (1983) Pm.Natl.Acad.Sci.USA 80,1646-1649. 11. Woodgett. J. & Hunter, T. (1987) J.Biol.Ckm. 262,48364843. 12. Kikkawa, U.. Minakuchi. R., Takai, Y.& Nishizuka, Y.(1983) Meth.Enzymol. 99,288-298. 13. Bradford, M. (1976) Anal.Biochem. 72.248-254. 14. Laemmli, U. (1970) Nature 227,680-685. 15. Pearce. F., Banks. B.. Banthorpe. D.. Davies. H. & Vernon. C. (1972) Eur.J.Biochem.29,4 17425. 16. Kosaka. Y.,Ogita. K., Ase, K., Nomura, H.,Kikkawa, U. & 151,973Nishizuka. Y.(1988) Biochern.Biophys.Rcs.Cornmun. 981.
3 . .
catalyzes the incorporation of 1.O nmoVmin phosphate into histone at
Step
The apparent activation constant for Ca2+ was 0.4pM; the K,,,s for ATP and histone were 8pM and 43pg/ml respectively. 'Ihe unsaturated fatty acids, arachidonic acid, oleic acid and linoleic acid, also activated mammary PKC to a smaller extent, but this activation was independent of the presence of phosphatidylserine and diolein. Divalent metal ions (at 5mM) had different effects on PKC activity; Mg2+ activated, MII~+activated very slightly and ~ n 2 +inhibited completely. Of several potential inhibitors tested,only staumporine was completely effective, H-7 less so. and both quercetin and gossypol were inactive. A study of the substrate specificity indicated that histone was better than casein. In summary. our results indicate that PKC from rat mammary gland behaves similarly to PKCs from other tissues. Funher studies of the regulation of mammary PKC by hormones and growth factors will improve our understanding of the complex process of mammary gland growth and development.
Isolation and biochemical characterization of protein kinase C from rat mammary gland S.LAVANDERO. E.MONCADA and M. SAPAG-HAGAR Departmento de Bioqufmica y Biologfa Molecular, Facultad de Ciencias Qufmicas y Famduticas. Universidad de Chile, Casilla 233, Santiago, Chile.
Protein phosphorylation is a prominent mechanism by which hormones and growth factors modulate cellular functions[1,2]. One of the major transduction pathways, receptor-mediated hydrolysis of phospholipids, leads to the activation of protein kinase C (PKC)[3,4]. PKC is widely distributed and its activity is diacylglycerol-. phospholipid-. and calcium- dependent[5]. Accumulating evidence, both direct and indirect, indicates that PKC plays an imponant role in the development of mammary tissue[6-10]. Mouse mammary PKC activity shows developmentrelated regulation. a decrease being observed during pregnancy and throughout lactation[6]. Because of the role of PKC in mammary growth and differentiation, we have purified and characterized the enzyme from this tissue. Mammary PKC was purified from Sprague-Dawley rats in midpregnancy, the stage at which activity is maximal (Lavandero. S.. Moncada, E.,Foncea, R., and Sapag-Hagar, M.. unpublished work). by a three-step procedure essentially as described by Woodgea er al.[ll]. The PKC activity was measured by the incorporation of 32P from [32P]ATP into Hl-histone[l2]. Protein concentrations were determined by the Bradford method[l3]. Sodium dodecyl sulphatepolyacrylamide electrophoresis and isoelectric focusing were performed as described previously[l4.15). On DEAE column chromatography. PKC eluted as a peak in the first fractions at 20-100mM-NaO. On Phenyl Sepharose. the enzyme eluted as a sharp peak at an ammonium sulphate Affinity chromatography using concentration close to zero. phosphatidylserine-acrylamide yielded a sharp peak of activity. Table 1 summarizes the purification data. Silver staining of polyacrylamide gels containing sodium dodecyl sulphate showed a protein band with an apparent molecular weight of 78kDa; isolectric focusing gave two bands with isoelectric points of 5.8 and 5.9. Both values agree with those obtained for PKC from other sources[3J.16]. Because of the instability of this preparation, however, partiallypurified enzyme was used in the subsequent biochemical characterization. Mammary PKC activity was dependent on phosphatidylserine and Ca2+and showed a marked concentrationdependent increase in the presence of diolein or 12-0-tetradecanoylphorbol 13-acetate. Table 1.
One unit (U) of activity is defined as that amount of PKC that 27°C. Volume
Rotcin conc.
Total Activity
Specific Activity
Purification
Yield
(ml)
(mg/ml)
(V)
(U/mg)
(fold)
(%)
Extract
14
9.03
0.81
0.01
DEAE cellulose
45
1.12
12.83
0.25
I .o
100
Phenyl Sepharose
65
0.21
11.05
0.81
3.2
86
PS-anyla-
2
0.02
0.06
1.40
5.5
4
mi&
We are indebted to Dr. C.I. Fogson for his help in the preparation of this manuscript. 'Ihis work was supported by Grants Fondecyt 91-0879 and DTI Universidad de Chile B-3113-9013. S.L. is a recipient of a fellowship from Fundaci6n Andes. 1. Nestler, E.& Greengard. P. (1984) Science 225.1357-1364. 2. Hunter, T. &Cooper. J.A. (1985) Annu.Rev.Biochem. 54,897930. 3. Nishizuka, Y.(1988) Nature 334,661-665. 4. Benidge, M.J. (1987) Annu.Rev.Biochem. 56,159-193. 5. Nishizuka, Y.(1986) Science 233,305-312. 6. Caulfield, J.J. & Bolander, F.F. (1986) J.Endocrino1.109,29-34. 7. Katoh, N. (1990) J.Dairy Sci. 73,1201-1207. 8. Holladay. C.S. & Bolander, F.F. (1986) Pm.Soc.Exp.Biol.Mcd. 183,343-347. 9. Waters, S.B. & Rillema, J.A. (1989) Mol.Cell.Endocrinol. 63.159-166. 10. Taketani, Y.& Oka, T. (1983) Pm.Natl.Acad.Sci.USA 80,1646-1649. 11. Woodgett. J. & Hunter, T. (1987) J.Biol.Ckm. 262,48364843. 12. Kikkawa, U.. Minakuchi. R., Takai, Y.& Nishizuka, Y.(1983) Meth.Enzymol. 99,288-298. 13. Bradford, M. (1976) Anal.Biochem. 72.248-254. 14. Laemmli, U. (1970) Nature 227,680-685. 15. Pearce. F., Banks. B.. Banthorpe. D.. Davies. H. & Vernon. C. (1972) Eur.J.Biochem.29,4 17425. 16. Kosaka. Y.,Ogita. K., Ase, K., Nomura, H.,Kikkawa, U. & 151,973Nishizuka. Y.(1988) Biochern.Biophys.Rcs.Cornmun. 981.
3 . .
catalyzes the incorporation of 1.O nmoVmin phosphate into histone at
Step
The apparent activation constant for Ca2+ was 0.4pM; the K,,,s for ATP and histone were 8pM and 43pg/ml respectively. 'Ihe unsaturated fatty acids, arachidonic acid, oleic acid and linoleic acid, also activated mammary PKC to a smaller extent, but this activation was independent of the presence of phosphatidylserine and diolein. Divalent metal ions (at 5mM) had different effects on PKC activity; Mg2+ activated, MII~+activated very slightly and ~ n 2 +inhibited completely. Of several potential inhibitors tested,only staumporine was completely effective, H-7 less so. and both quercetin and gossypol were inactive. A study of the substrate specificity indicated that histone was better than casein. In summary. our results indicate that PKC from rat mammary gland behaves similarly to PKCs from other tissues. Funher studies of the regulation of mammary PKC by hormones and growth factors will improve our understanding of the complex process of mammary gland growth and development.
