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Phosphorylation and dephosphorylation of proteins in breast cancer cells
150
P285
Cell Biology
International
PHOSPHORYLATIONAND DEPHOSPHORYLATION OF PROTEINS
Reports,
P288
Kate Hammond, Marjorie Petersen. Department of Medical Biochemistry, University of the Witwatersrand Medical School, Johannesburg, South Africa. Protein phosphorylation and dephosphorylation, resulting from the action of kinases and phosphatases, respectively, has been studied in MCF7 human breast cancer cells. Cell extracts were prepared and incorporation of ['*PI-ATP into endogenous proteins was assessed in the absence or presence of kinase or phosphatase modulators; proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and visualised by autoradiography. A number of major bands were detected and changes in distribution, which may be of importance in cancer cell function, were seen with certain activators and inhibitors. Electrophoretic studies of the various forms of protein kinases in the cell preparations were also carried out; in this case nondenaturing polyacrylamide gels were used. After electrophoresis, the gels were incubated with ["PI-ATP in the absence or presence of histone substrate and activity was detected by autoradiography. Several major bands of histone-dependent kinase activity were seen; the faster moving forms were able to autophosphorylate. The different kinases observed may have an essential role in cell metabolism and studies aimed at elucidating their nature and function are reouired.
PROTEIN KINASE C IN ISCHEMICREPERFUSED LIVER CELLS R. PICCOLBTTI, P. BENDINBLLI, D. ARIENTI and A. BERNELLI-ZAZZERA Istituto di Patologia Generale dell'universiti degli Studi; Centro di Studio sulla Patologia Cellulare 31 - 20133 MILAN0 (Italy). de1 CNR - Via Mangiagalli
P287
Activity and sub-cellular distribution of protein kinase C(PKC) were studied in rat livers during tern2 orary non-necrogenic ischemia and post-ischemic reperfusion. Interruption of blood supply for 60 min caused a significant decrease of PKC activity in both membrane (-50%) and cytosol (-40%) fractions. Recovery of PKC activity was very slow during reper fusion and normal levels were attained only after 10 h from the re-establishement of blood supply. Di& cylglycerol (DAG), an endogenous activator of PKC, increased in amount in ischemic livers, and the increase was intensified during blood reperfusion. To tal P-lipid, and defined species such as phosphatidyl-ethanolamine and phosphatidyl-choline, decreased in amxnt during ischemia, and returned to baseline levels within 1 h after reperfusion. Changes in DAG and P-lipid concentrations during ischemia are consistent with activation of endogeneous phospholipases, while the impossibility to demonstrate changes in Ca2* and phospholipid-independent PKC activity in the cytosol argues against a possible role of DAG in the down-regulation of PKC in ischemic and postischemic livers. Upon reperfusion, hormonal factors insulin) might play a role in increasing DAG (e.g., concentration: the long time period necessary for restoration of PKC activity might indicate that pro tein itself was damaged during ischemia and synthesis of new molecules is necessary for recovery of activity. Immunoblot studies are in progress to demonstrate this hypothesis.
-ULAR
FOR
Supplement
CLONlNGOFTHEHUMAN SuBuNlT
A REGULAl7IRY OFcAMP~PRoTELNKIN~ cDNA
IN BREAST CANCER CELLS
Vol. 74, Abstracts
1990
(RI@
Rinmor
Solberg Anne Keiserud and Tore Jahnsen. for &me Technology, Institute of Pathology, Rikshospitalet, N-0027 Oslo 1; and Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo 3; Laboratory Norway. Many hormones exert their effects on cellular metabolism and gene expression by regulating CAMP levels and thereby the activity of the cAh4P-dependent protein kinase (PKA). The PKA holoenzyme consists of two regulatory (R) and two catalytic (C) subunits. Two major isozymes of PKA, distinguished by the R subunits (designated RI and RII), have been described. Multiple isoforms of both the R and C subunits, designated RIu, RI& RIIo, RII/3, Cu, Cfi and C7, have been identified at the gene/mRNA level. We have isolated three cDNA clones of 2.4 kb, 2.1 kb and 1.6 kb, respectively, for Rv from a human testis cDNA library, using the corresponding mouse cDNA as a probe. Nucleotide sequencing showed all three clones to represent partial clones for human RI& The clone of 2.4 kb was almost a full-length clone starting at the G in the ATG start codon, compared to mouse RIB. Computer studies of the similarity at the nucleotide level showed about 65 % to mouse RI& 75 % to mouse RIa and 73 96 to human RP, respectively, in the coding region. We are presently isolating the 5’ end of this cDNA using the PCR (Polymerase Chain Reaction) technique.
P285
Cell Biology
International
PHOSPHORYLATIONAND DEPHOSPHORYLATION OF PROTEINS
Reports,
P288
Kate Hammond, Marjorie Petersen. Department of Medical Biochemistry, University of the Witwatersrand Medical School, Johannesburg, South Africa. Protein phosphorylation and dephosphorylation, resulting from the action of kinases and phosphatases, respectively, has been studied in MCF7 human breast cancer cells. Cell extracts were prepared and incorporation of ['*PI-ATP into endogenous proteins was assessed in the absence or presence of kinase or phosphatase modulators; proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and visualised by autoradiography. A number of major bands were detected and changes in distribution, which may be of importance in cancer cell function, were seen with certain activators and inhibitors. Electrophoretic studies of the various forms of protein kinases in the cell preparations were also carried out; in this case nondenaturing polyacrylamide gels were used. After electrophoresis, the gels were incubated with ["PI-ATP in the absence or presence of histone substrate and activity was detected by autoradiography. Several major bands of histone-dependent kinase activity were seen; the faster moving forms were able to autophosphorylate. The different kinases observed may have an essential role in cell metabolism and studies aimed at elucidating their nature and function are reouired.
PROTEIN KINASE C IN ISCHEMICREPERFUSED LIVER CELLS R. PICCOLBTTI, P. BENDINBLLI, D. ARIENTI and A. BERNELLI-ZAZZERA Istituto di Patologia Generale dell'universiti degli Studi; Centro di Studio sulla Patologia Cellulare 31 - 20133 MILAN0 (Italy). de1 CNR - Via Mangiagalli
P287
Activity and sub-cellular distribution of protein kinase C(PKC) were studied in rat livers during tern2 orary non-necrogenic ischemia and post-ischemic reperfusion. Interruption of blood supply for 60 min caused a significant decrease of PKC activity in both membrane (-50%) and cytosol (-40%) fractions. Recovery of PKC activity was very slow during reper fusion and normal levels were attained only after 10 h from the re-establishement of blood supply. Di& cylglycerol (DAG), an endogenous activator of PKC, increased in amount in ischemic livers, and the increase was intensified during blood reperfusion. To tal P-lipid, and defined species such as phosphatidyl-ethanolamine and phosphatidyl-choline, decreased in amxnt during ischemia, and returned to baseline levels within 1 h after reperfusion. Changes in DAG and P-lipid concentrations during ischemia are consistent with activation of endogeneous phospholipases, while the impossibility to demonstrate changes in Ca2* and phospholipid-independent PKC activity in the cytosol argues against a possible role of DAG in the down-regulation of PKC in ischemic and postischemic livers. Upon reperfusion, hormonal factors insulin) might play a role in increasing DAG (e.g., concentration: the long time period necessary for restoration of PKC activity might indicate that pro tein itself was damaged during ischemia and synthesis of new molecules is necessary for recovery of activity. Immunoblot studies are in progress to demonstrate this hypothesis.
-ULAR
FOR
Supplement
CLONlNGOFTHEHUMAN SuBuNlT
A REGULAl7IRY OFcAMP~PRoTELNKIN~ cDNA
IN BREAST CANCER CELLS
Vol. 74, Abstracts
1990
(RI@
Rinmor
Solberg Anne Keiserud and Tore Jahnsen. for &me Technology, Institute of Pathology, Rikshospitalet, N-0027 Oslo 1; and Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo 3; Laboratory Norway. Many hormones exert their effects on cellular metabolism and gene expression by regulating CAMP levels and thereby the activity of the cAh4P-dependent protein kinase (PKA). The PKA holoenzyme consists of two regulatory (R) and two catalytic (C) subunits. Two major isozymes of PKA, distinguished by the R subunits (designated RI and RII), have been described. Multiple isoforms of both the R and C subunits, designated RIu, RI& RIIo, RII/3, Cu, Cfi and C7, have been identified at the gene/mRNA level. We have isolated three cDNA clones of 2.4 kb, 2.1 kb and 1.6 kb, respectively, for Rv from a human testis cDNA library, using the corresponding mouse cDNA as a probe. Nucleotide sequencing showed all three clones to represent partial clones for human RI& The clone of 2.4 kb was almost a full-length clone starting at the G in the ATG start codon, compared to mouse RIB. Computer studies of the similarity at the nucleotide level showed about 65 % to mouse RI& 75 % to mouse RIa and 73 96 to human RP, respectively, in the coding region. We are presently isolating the 5’ end of this cDNA using the PCR (Polymerase Chain Reaction) technique.