Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast‐atom‐bombardment mass spectrometry

Abstract

The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the /3-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and Cterminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28 -C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28 -C39 from 0.7 to 1.2 mol/mol and ClOO from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin.Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28 -C39, C46, C87 and ClOO in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed.Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.Glycogen synthase, the rate-limiting enzyme in glycogen synthesis, is regulated by multisite phosphorylation. Up to 1983, seven phosphorylation sites had been identified in the rabbit skeletal muscle enzyme, all located in the amino-terminal (N) or carboxy-terminal (C) cyanogen bromide peptides [l, 21. The amino acid sequences of these regions are shown in Fig. 1. In the N-terminal domain, residue N7 was phosphorylated in vitro by the calmodulin-dependent multiprotein kinase [3 -51, phosphorylase kinase [6,7], cyclic-AMP-dependent protein kinase [8] and glycogen synthase kinase-4 [9]. In the C-terminal domain, glycogen synthase kinase-3 phosphorylated three residues C30, C34 and C38, all contained within the same tryptic peptide [lo], while casein kinase-11 labelled residue C46 [9]. Cyclic-AMP-dependent protein kinase phosphorylated residues C87 and ClOO [8], while C100 was also a target for the calmodulin-dependent multiprotein kinase [3, 41. The in vivo phosphorylation state of glycogen synthase is under hormonal control, increasing in response to adrenalin (decreasing activity) and decreasing in response to insulin (increasing activity) [l 11. In normally fed animals injected with the P-adrenergic antagonist propranolol, the alkali-labile phosphate bound covalently to glycogen synthase was found to be 2.9 mol/inol subunit and this value increased to 5.1 mol/ mol subunit in animals injected with adrenalin [12]. In animals deprived of food for ...

The molecular mechanism by which adrenalin inhibits glycogen synthesis

“…The heat-treated enzyme was therefore stored at 4°C. In calculating phosphorylation stoichiometries, the apparent molecular mass of the glycogen synthase (86 kDa)/glycogenin (38 kDa) complex was taken as 124 kDa and protein was determined according to Bradford Glycogen synthase was also isolated in the presence of NaF from rabbits that had been injected with either adrenalin or propranolol as described [2,31. These preparations were made by Dr Julie Pitcher in this laboratory.…”

“…The activity ratio is approximately 0.2 in normally fed rabbits injected with propranolol, decreasing to about 0.03 following administration of adrenalin [3]. It is established that phosphorylation of residues C42 [2], C46 [4, 51 and ClOO [6] in vitro has no effect, and C87 very little effect [6], on the activity ratio. N7 [S], and especially C30-C38 [5,71 are the sites whose phosphorylation inhibits glycogen synthase in vitro, and their effects are additive, greater de- creases in the activity ratio being observed when both regions are phosphorylated [8].…”

“…However, N10 is only one of about 10 serine residues phosphorylated by CKI in vitro [9], several of which are not phosphorylated significantly in vivo [2]. Although these observations initially suggested that CK1 might not be the protein kinase responsible for the phosphorylation of N10 in vivo, Roach and coworkers [lo] recently reported that if glycogen synthase was first phosphorylated by cyclic-AMP-dependent protein kinase (PKA), it became a better substrate for CKI.…”

Yeast casein kinase I homologues: an essential gene pair.

“…However, the importance of phosphorylation by the enzyme in vivo has been demonstrated in few cases, notably glycogen synthase. Phosphorylation of glycogen synthase by casein kinase I in vitro results in inactivation of the enzyme (7) and phosphorylation of one site, Ser10, occurs in rabbit muscle in vivo in response to stimulation by epinephrine (9). Other physiologically important phosphorylations by casein kinase I have been suggested but its overall physiological roles remain unclear.…”

Caracterização molecular da proteína DdI-2 e mapeamento de seus domínios de interação com a proteína fosfatase do tipo-1 de Dictyostelium discoideum

“…O I-1 está envolvido em diversos processos celulares como na sinapse controlada por neurotransmissores, o controle de células tumorais, o controle da contração muscular e o controle hormonal do metabolismo de glicogênio (Oliver & Shenolikar, 1998 (Poulter et al, 1988;Nakielny et al, 1991;Oliver & Shenolikar, 1998 (Scrimgeour et al, 1999;Zheng et al, 2000).…”