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

Abstract

1. The effects of glucagon, insulin and phenylephrine on the phosphorylation of cytoplasmic, mitochondrial and membrane proteins were studied in intact hepatocytes from 24 h-starved rats incubated with [32P]Pi. A rapid cell-fractionation technique was used, followed by radioautography of the proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. Glucagon consistently caused a significant increase in the phosphorylation of four readily separable cytoplasmic phosphoproteins, of Mr 93000, 50000, 46000 and 20000, and a decrease in phosphorylation of a phosphoprotein of Mr 22000. Phosphorylation of the protein of Mr 46000 was also enhanced by both phenylephrine and insulin, and that of Mr 93000 by phenylephrine. 3. The phosphoprotein of Mr 22000 was not precipitated by boiling for 5 min, and had a mobility identical with that of similar protein whose phosphorylation is enhanced in the adipocyte by insulin [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383]. 4. Glucagon, but not phenylephrine or insulin, enhanced the phosphorylation of a mitochondrial protein of Mr 35000 and of four plasma- or microsomal-membrane proteins of Mr 50000, 30000, 23000 and 19000. 5. Mitochondria from glucagon-treated animals or hepatocytes phosphorylated a protein of Mr 30000 when incubated in vitro with [32P]Pi and ADP. Phosphorylation of this protein did not occur with mitochondria from control, phenylephrine- or insulin-treated cells. 6. The significance of these hormonally induced changes in protein phosphorylation is discussed.

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

“…4, lane b) and forskolin (Fig. 4, lane c) stimulated the phosphorylation of various proteins in isolated rat hepatocytes, among them a weakly phosphorylated 23-kDa protein which is most likely the phosphoprotein described by Vargas et al [53]. We were, however, unable to detect under these conditions a phosphoprotein with the same apparent molecular size as protein 111.…”

“…phospholamban) [13 -191, platelets [26 -451, liver [53], adrenal cortex [63,64], adipocytes [65,66], granulocytes [67], Leydig tumor cells [69], bovine lens [70, 711 and in 3T3 cells [72]. However, in most cases the possible identity of these proteins has not been explored.…”

“…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. We can show here that the 23-kDa phosphoproteins from platelets and hepatocytes are distinct from protein I11 found in microsomal membranes from parotid glands and other exocrine organs.…”

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

“…When isolated rat liver mitochondria were incubated with g 32 P-ATP, phosphoproteins of 32 and 42 kDa were detected and identified as two different forms of succinyl-CoA synthetase [7]. Additionally, a 35-kDa mitochondrial protein was shown to be phosphorylated in rat hepatocytes in response to glucagon; the identity of the mitochondrial protein kinase responsible for the phosphorylation of the 35-kDa protein, however, was not known [8]. These studies showed that mitochondria contained enzymes capable of catalysing protein phosphorylation.…”

cAMP‐dependent protein phosphorylation in mitochondria of bovine heart

“…Glucacon also stimulates phosphorylation of mitochondrial proteins in vivo [4] or in isolated cell preparations [5]. CAMP itself seems to affect mitochondrial processes [6], and CAMP binding proteins have been detected in mitochondria [7,8].…”