The glycogen-binding subunit of protein phosphatase-1g from rabbit skeletal muscle

Further characterisation of its structure and glycogen-binding properties

Authors


Correspondence to P. Cohen, Department of Biochemistry, Medical Sciences Institute, The University, Dundee DD1 4HN, Scotland

Abstract

The glycogen-bound form of protein phosphatase-1 (PP-1g) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G′) is itself a product of proteolysis.

A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation.

PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.

Abbreviations
PP-1G

glycogen-bound form of protein phosphatase-1

A-kinase

cAMP-dependent protein kinase

site 1

Ser-Pro-Gln-Pro-Ser-Arg-Arg-Gly-Ser-Glu-Ser-Ser-Glu-Glu

PhMeSo2F

phenylmethanesulphonyl fluoride

TosPheCH2Cl

tosylphenyl-chloromethane

Enzymes
 

Protein phosphatase-1 (EC 3.1.3.16)

 

cAMP-dependent protein kinase (EC 2.7.1.37)

 

trypsin (EC 3.4.21.4)

 

α-amylase (EC 3.2.1.1)

 

phosphorylase kinase (EC 2.7.1.38)

 

glycogen synthase (EC 2.4.1.11)

 

glycogen phosphorylase (EC 2.4.1.1)

 

glycogen debranching enzyme (EC 3.2.1.33 and EC 2.4.1.25)

Ancillary