Effects of phosphorylation of protein phosphatase 1 by pp60v-src on the interaction of the enzyme with substrates and inhibitor proteins

• Published in: Biochimica et biophysica acta Vol. 928; no. 1; p. 63
• Main Authors: Johansen, J W, Ingebritsen, T S
• Format: Journal Article
• Language: English
• Published: Netherlands 02-04-1987
• Subjects: Adenosine Triphosphate - metabolism; Chymotrypsin; Glycogen Synthase - metabolism; Kinetics; Oncogene Proteins, Viral - metabolism; Peptide Fragments - metabolism; Phosphoprotein Phosphatases - metabolism; Phosphoproteins - metabolism; Phosphorylase Kinase - metabolism; Phosphorylases - metabolism; Phosphorylation; Protein Phosphatase 1; Protein-Tyrosine Kinases - metabolism; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0006-3002
• DOI: 10.1016/0167-4889(87)90086-3

Phosphorylation of protein phosphatase 1 by pp60v-src decreased its activity towards phosphorylase kinase and glycogen synthase as well as towards phosphorylase a. Kinetic experiments indicated that the primary effect of phosphorylation was to increase the Km for each of the substrate proteins. There was little or no change in the Vmax for the reactions. The possibility that phosphorylation of protein phosphatase 1 altered its regulation by inhibitors-1 and -2 was also examined. Phosphorylation of protein phosphatase 1 did not prevent the reversible inhibition of the enzyme by inhibitor-1 or inhibitor-2 nor did it prevent the association of inhibitor-2 with protein phosphatase 1 to form the MgATP-dependent protein phosphatase. Protein phosphatase 1 is not a substrate for pp60v-src when it is complexed with inhibitor-2 to form the inactive MgATP-dependent protein phosphatase. Here we have shown that protein phosphatase 1 is also not phosphorylated by pp60v-src following activation of the MgATP-dependent protein phosphatase with glycogen synthase kinase-3 and MgATP. This indicates that the inability of pp60v-src to phosphorylate protein phosphatase 1 is not due to the change in protein phosphatase 1 conformation which accompanies the inactivation of the MgATP-dependent protein phosphatase. Rather, it appears to be the result of steric hindrance by inhibitor-2. This suggests that the pp60v-src phosphorylation site is closely associated with the inhibitor-2 binding site involved in the formation of the MgATP dependent protein phosphatase. The pp60v-src phosphorylation site was previously localized to a small (Mr less than or equal to 4000) domain which can be selectively degraded by chymotrypsin. Here we have shown that chymotryptic digestion increased the Km of unphosphorylated protein phosphatase 1 for each of the three phosphoprotein substrates used in this study. This effect was similar to that observed after phosphorylation of protein phosphatase 1. These results indicate that the pp60v-src phosphorylation site is in a region of protein phosphatase 1 which influences substrate binding and which may be near the active site.