Interaction of p130 with, and Consequent Inhibition of, the Catalytic Subunit of Protein Phosphatase 1α

Abstract

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-␦1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1␣ (PP1c␣) as a p130-binding protein. The association between p130 and PP1c␣ was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1c␣ resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1c␣ also detected the presence of a complex of p130 and PP1c␣. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1c␣, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca 2؉ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1c␣.D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ), 1 a product of receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) by phospholipase C (PLC), plays an important role as an intracellular second messenger by mobilizing Ca 2ϩ from nonmitochondrial stores (1). We previously isolated two Ins(1,4,5)P 3 -binding proteins with molecular masses of 130 and 85 kDa from rat brain (2, 3) with the use of an Ins(1,4,5)P 3 affinity column (4, 5). Partial amino acid sequencing revealed that the 85-kDa molecule was PLC-␦1 (2). Identification of the pleckstrin homology (PH) domain of PLC-␦1 as the site of Ins(1,4,5)P 3 binding helped to define the PH domain as an inositol compound binding module (6, 7). The Ins(1,4,5)P 3 -binding protein with a molecular mass of 130 kDa, termed p130, was a previously unidentified molecule (2, 3). The predicted amino acid sequence of rat p130 shares 38.2% identity with that of rat PLC-␦1; the five identified domains of PLC-␦1 (PH, EF-hand, putative catalytic (X and Y), and C2 domains) are all present in p130. The domain organization of p130 suggests that the protein is likely to possess a fold similar to that of PLC-␦1, a notion that is supported by the results of limited proteolysis with trypsin (8). However, p130 exhibits some distinct characteristics. It is larger than the PLC-␦ isozymes, and it possesses unique regions both at the NH 2 terminus, preceding the PH domain, and at the COOH terminus. Moreover, the residues within the catalytic domain of PLC-␦ that are critical for enzyme activity (His 356 and Glu 390 ) are not conserved in p130 (9). The PH domain of p130, like that of PLC-␦1, is important for the binding of Ins(1,4,5)P 3 (10). Other mol...

Phospholipase C-related but Catalytically Inactive Protein (PRIP) Modulates Synaptosomal-associated Protein 25 (SNAP-25) Phosphorylation and Exocytosis

“…3). PP1 caused the release of 32 P from phosphorylated SNAP-25, which was inhibited by the addition of purified recombinant PRIP-1 but not by the mutant PRIP-1, whose residue Phe-97 was replaced with Ala, lacking PP1 binding ability (29), as shown in Fig. 3A.…”

“…We isolated PRIP as a novel inositol 1,4,5-trisphosphate/PtdIns(4,5)P 2 -binding protein (23,32,34,35) and later identified it also as a protein phosphatase (PP1 and PP2A)-anchoring protein (29,30). Furthermore, very recently we found that PRIP associates with Akt when it is phosphorylated to be active (31), indicating that PRIP is a unique molecule participating in phosphorylation events from both sides.…”

“…Noteworthy findings were as follows: (i) dephosphorylation of WT by PP1 was almost double that of the mutant S28A/T29A, indicating that Ser-28/Thr-29 and Thr-138/Ser-187 are equally dephosphorylated by PP1; (ii) dephosphorylation of WT and the mutant S28A/T29A by PP2A were much the same, indicating that PP2A fails to dephosphorylate at Ser-28 and Thr-29; and (iii) the mutant S28A/T29A/T138A/ S187A was still dephosphorylated by PP1 and PP2A, indicating that unidentified residues corresponding to one or two residues are targets of PP1 and PP2A. (29). PRIP binds to PP1 to inhibit the phosphatase activity.…”

“…Further studies revealed that PRIP has a number of binding partners, including the catalytic subunit of protein phosphatase 1␣ (PP1␣) and PP2A (29,30) and the phosphorylated (active) form of Akt (31) in addition to Ins(1,4,5)P 3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) (32)(33)(34)(35)(36). Thus, PRIP is a unique molecule that associates with both multiple phosphatases (PP1 and PP2A) and a kinase (activated form of Akt), suggesting that PRIP participates in the phospho-regulation of cellular events, by recruiting these enzymes to where the event occurs if PRIP can approach.…”

GABAAReceptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

“…PRIP-1 has a number of binding partners, including catalytic subunit of protein phosphatase 1␣ (PP1␣) and GABA A receptor-associated protein (GABARAP) Yoshimura et al, 2001; Kanematsu et al, 2002). A role for PRIP-1 in regulating GABA A receptor function is further suggested by the phenotype of PRIP-1 knock-out (PRIP-1 Ϫ/Ϫ ) mice, which have altered GABA A receptor pharmacology and behavior .…”