Identification of Protein-Phosphatase-1-Binding Domains on the Glycogen and Myofibrillar Targetting Subunits

Authors


  • Note. D. F. Johnson, G. Moorhead and Y. H. Chen have all made equally important contributions to this study. D. F. Johnson studied the interaction of the M110 subunit and its fragments with PPIc G. Moorhead analysed the interaction of the GM subfragments with PPIc and Y. H. Chen made the M110 and GM constructs.

P. Cohen, MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland.
Fax:+44 382 223778.

Abstract

The specificity of the catalytic subunit of protein phosphatase-1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1C-binding domains on GL and GM the subunits that target PPIC, to hepatic and muscle glycogen, respectively, and on M110 the subunit that targets PP1C to smooth muscle myosin. GM-(G63–T93) interacted with PP1C and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1C or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1C-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM-(G63–N75) had the same effect as GM-(G63–T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1C because phosphate is inserted into the PP1C-binding domain of GM M110-(M1-E309) and M110-(M1-F38), but not M110-(D39–E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110-(M1-E309), neither M110-(M1-F38) nor M1110-(D39–E309) suppressed the PPC-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39–309 which contains seven ankyrin repeats. M110-(M1-F38) displaced GL from PPIC while GM-(G63-T93) displaced M110 from PP1Cin vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1C contain just a single targetting subunit.

Abbreviations
PP1

protein phosphatase-1

PP1c

catalytic subunit of PP1

PPlγ

γ isoform of PP1c

PP1G

glycogen-associated form of PP1

PP1M

myosin-associated form of PP1

GM

glycogen-binding subunit of PP1 from striated muscle

GL

glycogen-binding subunit of PP1 from liver

NIPP1

nuclear inhibitor of PP1

DARPP

dopamine-regulated and cyclic-AMP-regulated phosphoprotein

M21 and M110 myofibrillar-binding subunits of PP1 with molecular masses of 21 kDa and 110 kDa

respectively

PKA

cyclic-AMP-dependent protein kinase

PhMeSO2F

phenylmethylsulphonyl fluoride

GST

glutathione S -transferase

MLC20

myosin light chain of molecular mass 20 kDa

Ancillary