Protein Phosphorylation and Dephosphorylation in Liver Plasma Membrane. Effect of Inorganic Phosphate

Abstract

When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.

The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats

“…Blat & Loeb (1971) have reported that glucagon treatment of the whole rat enhanced the phosphorylation of a liver ribosomal protein of unspecified molecular weight. It has also been shown that isolated liver plasma membranes (Marchmont & Houslay, 1980;Kobayashi & Ozawa, 1981) or microsomal membranes (Behar-Bannelier & Murray, 1980) incubated with [y-32P]ATP and Mg2+ show many phosphorylated proteins on SDS/polyacrylamide-gel electrophoresis. Two of the plasma-membrane phosphoproteins, of Mr 23000 and 20000, show enhanced phosphorylation in the presence of 100mM-phosphate (Kobayashi & Ozawa, 1981).…”

“…It has also been shown that isolated liver plasma membranes (Marchmont & Houslay, 1980;Kobayashi & Ozawa, 1981) or microsomal membranes (Behar-Bannelier & Murray, 1980) incubated with [y-32P]ATP and Mg2+ show many phosphorylated proteins on SDS/polyacrylamide-gel electrophoresis. Two of the plasma-membrane phosphoproteins, of Mr 23000 and 20000, show enhanced phosphorylation in the presence of 100mM-phosphate (Kobayashi & Ozawa, 1981). It is possible that these correspond to the phosphoproteins of Mr 23 000 and 19 000 seen in the present experiments.…”

cAMP‐dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver

“…For hepatocytes, studies concerning hormone-induced protein phosphorylation have been largely restricted to cytosolic proteins [47 -511. Only a few reports deal with the phosphorylation of membrane proteins: Kobayashi and Ozawa [52] showed that a 23-kDa protein was phosphorylated in plasma membrane preparations. Vargas et al…”

“…For hepatocytes, studies concerning hormone-induced protein phosphorylation have been largely restricted to cytosolic proteins [47 -511. Only a few reports deal with the phosphorylation of membrane proteins: Kobayashi and Ozawa [52] showed that a 23-kDa protein was phosphorylated in plasma membrane preparations. Vargas et al [53] described a glucagon-induced phosphorylation of a 23-kDa membrane protein and postulated a relationship between this effect and the known action of glucagon on the ruthenium-red-insensitive transport of Ca2 The description of proteins of similar molecular size (22 -23 kDa) in microsomal membranbes from parotid acinar cells, from platelets and from hepatocytes which could all be phosphorylated in the intact cell by CAMP-dependent protein kinases, prompted us to study their possible identity.…”