The protein phosphatases involved in cellular regulation

Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle

Authors


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

Abstract

A type-1 protein phosphatase (protein phosphatase-1G) was purified to homogeneity from the glycogenprotein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase-1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel-filtration on Sephadex G-100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37-kDa type-1 protein phosphatase catalytic subunit (protein phosphatase-1C) isolated from ethanol-treated muscle extracts, as judged by peptide mapping. The G component was the glycogen-binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels.

Protein phosphatase-1G was inhibited by inhibitor-1 and inhibitor-2, but unlike protein phosphatase-1c, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase-1G was inactivated very rapidly by inhibitor-1. Protein phosphatase-1G interacted with inhibitor-2 (1–2) to form an inactive species, with the structure GCI-2. This form could be activated by preincubation with Mg-ATP and glycogen synthase kinase-3.

The G subunit could be phosphorylated on a serine residue(s) by cyclic-AMP-dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase-3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase-1G by inhibitor-1. The relationship of the G subunit to the ‘deinhibitor protein’ is discussed.

Abbreviation
SDS

sodium dodecyl sulphate

Enzymes
 

Glycogen phosphorylase (EC 2.4.1.1)

 

glycogen synthase (EC 2.4.1.11)

 

phosphorylase kinase (EC 2.7.1.38)

 

cyclic-AMP-dependent protein kinase (EC 2.7.1.37)

 

glycogen synthase kinase-3 (EC 2.7.1.37)

 

protein phosphatase-1 (EC 3.1.3.16)

 

trypsin (EC 3.4.21.4)

 

chymotrypsin (EC 3.4.21.1)

 

staphylococcal V8 proteinase (EC 3.4.21.19)

 

α-amylase (EC 3.2.1.1)

Ancillary