Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation

Advances in Biological Regulation xxx (2013) 1–10

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Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation Goro Sugiyama a, Hiroshi Takeuchi b, Takashi Kanematsu c, Jing Gao a, Miho Matsuda a, Masato Hirata a, * a

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan b Department of Applied Pharmacology, Kyushu Dental University, Kyushu University, Fukuoka 812-8582, Japan c Department of Cellular and Molecular Pharmacology, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima City, Japan

a b s t r a c t PRIP, phospholipase C (PLC)-related but catalytically inactive protein is a protein with a domain organization similar to PLC-d1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatase 1 and 2A (PP1c and PP2Ac), depending on the phosphorylation of PRIP. We also found that Akt was precipitated along with PRIP by anti-PRIP antibody from neuronal cells. In this article, we summarize our current reach regarding the interaction of PRIP with Akt and protein phosphatases, in relation to the cellular phospho-regulations. PP1 and PP2A are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled-down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but in close proximity. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the reduced binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the increased binding of PP2A in in vitro

* Corresponding author. Tel.: þ81 92 642 6317; fax: þ81 92 642 6322. E-mail address: [email protected] (M. Hirata). 2212-4926/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbior.2013.07.001

Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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experiments. This binding regulation of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling. Ó 2013 Elsevier Ltd. All rights reserved.

Introductiondwhat is PRIP? We described for the first time the chemical modification of inositol 1,4,5-trisphosphate [Ins(1,4,5) P3] (Hirata et al., 1985). The analog has the azidobenzoyl group at the C-2 position for photoaffinity labeling and causes irreversible inactivation of the receptor protein for Ca2þ release, following photolysis. Irvine et al. (1984) reported that the biological activity of Ins(1,4,5)P3 is related to two adjacent phosphates at C-4 and C-5 and that the phosphate at C-1 increases the affinity of its recognition by the receptor site. On the basis of this report and our findings, we attempted further chemical modification of Ins(1,4,5)P3 at the C-2 position and examined the effects on Ins(1,4,5)P3 recognizing molecules including the receptor and metabolizing enzymes in order to explore the recognition modes by these proteins (Hirata et al., 1989, 1990a, 1993, 1994). In the extension of this project, we also synthesized Ins(1,4,5)P3-immobilized beads, which proved to be useful for purifying the known Ins(1,4,5)P3-interacting molecules (Hirata et al., 1990b). When rat brain cytosol fraction was applied to this affinity column, we became aware of the presence of remarkable Ins(1,4,5)P3 binding activity in a high-salt eluate. Samples from the Ins(1,4,5) P3-column were further fractionated by applying them to a gel filtration column, dividing the binding activities into two peaks (130 kDa and 85 kDa proteins). Partial amino acid sequencing of these purified samples revealed that the 85 kDa molecule was phospholipase C-d1 (PLC-d1), while the protein with a molecular mass of 130 kDa, initially termed p130 based on its molecular size, was a previously unidentified protein (Kanematsu et al., 1992; Yoshida et al., 1994). Further studies on the region in PLC-d1 responsible for binding Ins(1,4,5)P3 have identified the pleckstrin homology (PH) domain as a specific structural module for accommodating inositol phosphates and phosphoinositides (Yagisawa et al., 1994, 1998; Lemmon et al., 1997; Hirata et al., 1998). Following studies to isolate the cDNA encoding p130 revealed its considerable similarity to phosphoinositide-specific PLC enzymes and, in particular, the PLC-d family (Kanematsu et al., 1996). Based on the crystal structure of PLC-d1 (Essen et al., 1996) and limited proteolysis experiments of p130 by trypsin (Kanematsu et al., 2000), p130 is predicted to have the same domain organization, incorporating the PH, EF-hand, catalytic (X and Y) and C2 domains in this order. However, p130 has some distinct characteristics. It is a larger molecule than the PLC-d isozymes and unique regions are present at both the N-terminus preceding the PH domain and at the C-terminus. More importantly, the residues within the catalytic domain critical for PLC activity (Glu341 and His356) are not conserved in p130 (Kanematsu et al., 1996). As expected from a mutagenesis study of PLC-d1 (Ellis et al., 1998), it has been found that p130 indeed lacks PLC activity (Kanematsu et al., 1992, 1996: Yoshida et al., 1994). Other molecules that show sequence similarity to p130, such as human PLC-L (Kohno et al., 1994) and the K10F12.3 gene product of Caenorhabditis elegans (Koyanagi et al., 1998), have also been described. Otsuki et al. (1999) later isolated a cDNA from the mouse brain that encodes a protein with 64% sequence identity to the entire PLC-L; they therefore termed this protein PLC-L2 and renamed the original PLC-L as PLC-L1. Furthermore, the gene for human type 2 p130 (KIAA1092) has also been cloned (Kikuno et al., 1999). All of these proteins exhibit characteristic NH2- and COOH-terminal extensions and replacement of critical catalytic residues such that the native or recombinant proteins encoded by these genes do not exhibit PLC activity (Otsuki et al., 1999; Koyanagi et al., 1998). The identification of a Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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p130-related molecule in such a simple organism as C. elegans suggests that this family of proteins diverged early from other PLC isozymes. However, the name of PLC is misleading because of the absence of catalytic activity. Therefore, we have proposed that this distinct family of PLC-related proteins is designated the PLC-related but catalytically inactive protein (PRIP) family (comprising PRIP-1 and -2 subfamilies) (see Fig. 1 for the domain organization). Binding partners of PRIP We applied the yeast two-hybrid system to identify proteins that interact with PRIP in order to explore further biological functions, because a variety of cellular signaling is mediated by proteinprotein interactions. With the unique NH2-terminal region of PRIP-1 as the bait for screening a human brain cDNA library, we isolated two positive clones, one of which was shown to encode the catalytic subunit of protein phosphatase 1a (PP1ac, Ref. Yoshimura et al., 2001). Another clone was found to be GABARAP (GABAA receptor associated protein) that was identified as a molecule capable of binding the g2 subunit of GABAA receptor and tubulin (Wang et al., 1999). In the process of experiments initiated by the finding of the above-mentioned binding partners, we also noticed that PRIP directly interacts with the b subunit of GABAA receptors (Terunuma et al., 2004) and the catalytic subunit of protein phosphatase 2A (PP2Ac) (Kanematsu et al., 2006). In addition, we also identified several other proteins including Akt (Fujii et al., 2010), syntaxin1 and SNAP-25 (synaptosomal-associated protein 25) (Zhang et al., 2013) as interacting proteins with PRIP. Fig. 2 depicts these interactions along with the amino acid residues responsible for the association. Phenotypes of PRIP gene-deficient mice The first trial to explore the functions of PRIP in cells was based on what PRIP was isolated as an Ins(1,4,5)P3 binding protein, indicating the implication of PRIP in the Ins(1,4,5)P3-mediated Ca2þ signaling pathway (Takeuchi et al., 2000; Harada et al., 2005). To explore further the biological functions of PRIP, we first generated PRIP-1 gene-deficient mice (PRIP-1 KO mice), followed by an analysis with a special reference to the function of interacting protein, GABARAP (Kanematsu et al., 2002). On the other hand, Takenaka et al. (2003) in a different laboratory generated PRIP-2 gene-deficient mice (PRIP-2 KO mice), whose phenotypes were seen in a B-cell receptor signaling pathway. By mating each genotype of the KO mice, we finally generated PRIP-1 and -2 double KO mice (DKO mice), followed by an extensive analysis from multiple functional aspects of mouse. In this article, however, we are not going to describe the phenotypes of these animals in detail, because of the limited space. Possible functions of PRIP in an animal itself and in primary cells there from, as assessed by analyzing DKO mice may be found in the following articles; we divide them into 5 categories: (1) a GABAA receptor function (Kanematsu et al., 2002, 2006, 2007; Terunuma et al., 2004; Yanagihori et al., 2006; Mizokami et al., 2007, 2010; Fujii et al., 2010; Migita et al., 2011; Kitayama et al., 2013); (2) an Ins(1,4,5)P3-mediated Ca2þ signaling pathway (Takeuchi et al., 2000, Harada et al., 2005); (3) a bone biology (Tsutsumi et al., 2011); (4) a reproduction system (Matsuda et al., 2009); (5) an exocytosis including insulin secretion (Doira et al., 2001, Gao et al., 2009, 2012; Zhang et al., 2013).

Fig. 1. Domain organization of PRIP-1: comparison with PLC-d1. Percentages indicate the amino acid identity in each domain of rat PRIP-1 compared with PLC-d1.

Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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Fig. 2. Binding partners of PRIP. Domain structures in PRIP are depicted with the number of the amino acid residues of PRIP-1 (11096). Molecules binding to PRIP are depicted along with the amino acid residues responsible for the interaction. The bars with numbers indicate the region responsible for the interaction with a respective molecule for PRIP-1. The numbers in the binding molecules indicate the region important for the interaction.

Mapping and the binding modes of PRIP to protein phosphatses Reversible phosphorylation is one of the most important post-translational modifications regulating protein characteristics, such as function and stability, especially those involved in cellular signaling. The importance of this modification is suggested by the fact that about one-third of all eukaryotic proteins are phosphorylated on specific serine, threonine, and/or tyrosine residues based on cellular status (Virshup and Shenolikar, 2009). Dynamic changes of the phosphorylation state of a protein result from the balance between localized activities of protein kinases and protein phosphatases, since each has a particular substrate preference (Cohen,1989). The phospho-states of a wide variety of substrate proteins need to be regulated in a spatio-temporal manner, which is brought about by the great diversity of regulatory proteins that form comparable numbers of holoenzymes; i.e., wide variety of regulatory proteins enables enzymes responsible for phospho-state of various substrate proteins with diverse substrate specificity by recruiting them to the vicinity of substrate proteins (Ceulemans and Bollen, 2004). As described above, our yeast two-hybrid analysis also revealed that PRIP interacts with PP1ac (Yoshimura et al., 2001; Uji et al., 2002), and further studies identified PP2Ac, and Akt as additional interacting proteins (Kanematsu et al., 2006; Fujii et al., 2010), indicating that PRIP might be implicated in phospho-regulation of several target proteins important for cellular functions. Despite the large numbers of regulatory subunits reported for PP1c and PP2Ac, there are only limited examples in which multiple phosphatases interact with a single molecule, for example, integrin aIIbb3 (Vijayan et al., 2004; Gushiken et al., 2008) and CG-NAP (centrosome and Golgi localized PKN-associated protein) (Takahashi et al., 1999). PRIP might be a member of these limited numbers of adaptor proteins. In addition, PRIP might be a special adaptor protein, since this is also able to bind to Akt, a serine/threonine kinase. To understand how these adaptor proteins serve multiple enzymes to target proteins for fine-tuning their phospho-states in a variety of cellular conditions, the relationship between an adaptor and each enzyme has to be investigated. PP1c interacts with PRIP-1 through the consensus binding motif for PP1c, 93KTVSF97 (Cohen, 1989), existing just at the amino-terminus to the PH domain of PRIP-1 (Yoshimura et al., 2001), and that PP2Ac also interacts with PRIP-1 through the region containing the PH domain (83-297 amino acids of PRIP-1) (Kanematsu et al., 2006), indicating that the sites responsible for binding are identical or very close. The PH domain of PRIP-1 immobilized on glutathione beads interacted with PP1c, which was displaced by recombinant PP2Ac protein in a dose-dependent manner. In reverse, the binding of PP2Ac to the beads increased dose-dependently. The results when performed vice versa, clearly indicate that the binding of Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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the catalytic subunits of both phosphatases to PRIP-1 is mutually exclusive in an either competitive or sterically exclusive manner. Phenylalanine at 97 of PRIP-1 is critical for accommodating PP1c, and thus the mutation to alanine (mutant F97A) diminished the binding, but no effect was seen with PP2Ac (Yoshimura et al., 2001; Terunuma et al., 2004; Sugiyama et al., 2012). No common sequences have been identified for binding to PP2Ac in the binding proteins so far reported. For instance, Yang et al. (2007) reported that PP2Ac interacts with Tap42/a4 via a region of this protein conserved among various species. They replaced positively charged residues Arg-163 and Lys-166 present in the conserved region (RxxKI, where ‘x’ indicates any residues) by reversing the charged residues, Glu and Asp, respectively (R163E/K166D), which resulted in reduced binding to PP2Ac. We found a similar sequence, 277KESKI281, in the region following the PH domain of PRIP-1 and therefore tested PP2Ac binding of mutated PRIP-1 (Lys-277 and Lys-280 were replaced by aspartate); however, there was no effect on PP2Ac binding. Integrin aIIb subunit was also shown to interact with the catalytic subunits of both PP1 and PP2A through a consensus motif, 989KVxF992, and a region, 989KVGFFKR995, respectively (Vijayan et al., 2004), indicating that the binding sites of PP1c and PP2Ac overlapped. We also found the presence of double basic amino acids (KK at 92 and 93 of PRIP-1) close to the consensus motif for PP1c; thus, we generated a mutant in which lysines at 92 and 93 were mutated to alanine and asparagine (K92A/K93N). The mutant K92A/ K93N robustly reduced the binding of PP2Ac, but nothing of PP1c, indicating that the amino acid residues responsible for the binding to PP1c and PP2Ac were different, but closely located. PP2Ac binding was also identified in another protein, Rb2/p130, by Purev et al. (2006). Two nuclear localization signals (1080SPSKRLRE and 1097TPTKKRGI) in the C-terminus of Rb2/p130 were both responsible for binding to PP2Ac, and therefore replacement of the basic residues within the motifs with alanine abrogated PP2Ac binding (Purev et al., 2011). Thus, we also examined whether the similar sequences present in PRIP-1 (101PSEKKISS and 149PSKKDLE) were involved in PP2Ac binding. Introduction of the mutations by replacing lysine residues with alanine or asparagine (K104A/K105N, K151N/K152A) resulted in the reduction of PP2Ac binding to PRIP-1. The basic residues mutated here (K151/K152) correspond to those for recognizing Ins(1,4,5)P3/ phosphatidylinositol 4,5-diphosphate by forming hydrogen bonds with the phosphate group of these inositol compounds (Ferguson et al., 1995). We therefore examined whether inositol phosphates and PP2Ac both affect the binding to PRIP-1. Neither 100-fold excess of Ins(1,4,5)P3 over PP2Ac nor 10-fold excess of PP2Ac over Ins(1,4,5)P3 showed an effect on the respective binding, indicating that PRIP-1 accommodates both Ins(1,4,5)P3 and PP2Ac simultaneously. In the structural model of the PH domain of PRIP-1 formed on the basis of that of PLC-d1, Ins(1,4,5)P3 appears to bind to the bottom surface that is formed by variable loops 1 and 3 in an analogy to the PH domain of PLC-d1 (Ferguson et al., 1995), while the binding to PP2Ac appears to be another surface of the PH domain composed of the variable loop 3 containing Lys-151/Lys-152 and the amino terminal extension to the PH domain containing Lys-92/Lys-93 and Lys-104/Lys-105. This might partially explain the lack of competition between PP2Ac and Ins(1,4,5)P3 for PRIP binding. It is also possible that the binding mode of PRIP for Ins(1,4,5)P3 is different from that of PLC-d1, although the binding affinity assayed in a solution using [3H]Ins(1,4,5)P3 was similar (Takeuchi et al., 1996). The model structure of the region of PRIP-1 for possible binding sites of Ins(1,4,5)P3, PP1c and PP2Ac is shown in Fig. 3. The effect of complex formation with PRIP-1 on the catalytic activities of PP2A, together with PP1 was analyzed, using [32P]phosphorylase or [32P]myosin light chain as a protein substrate. PRIP-1 dosedependently inhibited PP1c phosphatase activity, whereas it did not affect PP2A phosphatase activity at concentrations up to 100 molar excess of PRIP over the phosphatase (Yoshimura et al., 2001; Sugiyama et al., 2012). Modulation of the binding to protein phosphatases by the phosphorylation of PRIP The phosphorylation of PRIP-1 itself by PKA and PKC on the binding of PP1c and PP2Ac was analyzed; Thr-94 adjacent to the PP1c binding motif in PRIP-1 was phosphorylated by PKA both in vitro and in vivo, and the phosphorylation at Thr-94 no longer maintains PP1c binding (Terunuma et al., 2004; Gao et al., 2012; Sugiyama et al. 2012), resulting in the liberation of PP1c to be an active form. On the other hand, the binding of PP2Ac was not changed by the phosphorylation of PRIP, but in Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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Fig. 3. The model structure of the region of PRIP-1 for possible binding sites for Ins(1,4,5)P3, PP1c and PP2Ac. The 3D coordinates of the PH domain of PRIP-1 was generated with SWISS-MODEL server using the X-ray crystallographic structure of the PH domain of PLC-d1 (PDB code: 1MAI) as the template and visualized with PyMol software (http://www.pymol.org/). Adopted from Ref. Sugiyama et al. (2012).

reversely, the binding of PP1c and PP2Ac to PRIP-1 in a mutually exclusive manner led us to assume that the reduction of PP1c binding by the phosphorylation is accompanied by the increased binding of PP2Ac, i.e., phosphatses swapping. Indeed, PKA treatment of PRIP-1 increased PP2Ac binding, indicating that not only PP1c but also PP2Ac binding to PRIP is regulated by the phosphorylation of PRIP in cells. Thus, the vicinity of PRIP would be more active for dephosphorylating the surrounding substrate proteins following the phosphorylation at Thr-94, as shown in Fig. 4. Physiological relevance of the binding to protein phophatases, and the regulation by the phosphorylation In vitro binding experiments clearly indicated that PRIP-1 accommodates the catalytic subunits of PP1 and PP2A, and the binding is modified by phosphorylation of PRIP-1 itself. However, the results do not necessarily indicate that the binding and the modulation are of physiological relevance. Brain lysates contain intrinsic PP1c and PP2Ac, and the structural A subunit of PP2A (PP2Aa), as well as PRIP-1. The result clearly showed that PP1c and PP2Ac were co-precipitated with PRIP-1 only from the brain lysate of wild-type mice, but not of PRIP-DKO mice. From the band density in the precipitates compared to that in the lysates, as assessed by anti-PRIP-1 antibody, what about 9% of PRIP-1 present in the lysates is immunoprecipitated by anti-PRIP-1 antibody is estimated. Taking this value into account, a similar calculation based on band densities by each antibody led to the result that about 0.55% and 0.31% of PP1c and PP2Ac present in brain were co-precipitated with PRIP-1, respectively, whereas

Fig. 4. Schematic representation of regulatory modes of PP1c and PP2Ac by PRIP. (A) and (B) represent PRIP status in cells unstimulated and stimulated for phosphorylation, respectively. Phosphatases in round or serrated margins indicate inactive or active forms, respectively. Adopted from Refs. Terunuma et al. (2004), Gao et al. (2012), Sugiyama et al. (2012).

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PP2Aa appeared to be co-precipitated maximally up to 0.19% with PRIP-1, probably through the interaction with the catalytic subunit because the structural A subunit of PP2 itself is incapable of binding (Sugiyama et al., 2012). This rate difference between C and A subunit of PP2A indicated preferable binding of PRIP-1 to the monomeric catalytic subunit rather than the pre-formed AC core dimer of PP2A (PP2Aac). Given that there is as much PRIP-2 as PRIP-1, and type 2 also interacted with the catalytic subunits of PP1 and PP2A with similar affinity to PRIP-1 (Uji et al., 2002; Yanagihori et al., 2006), the above-mentioned percentage would be twice as much in the brain. Therefore, about 2% cellular PP1c and PP2Ac appears to form a complex with PRIP; this percentage is relatively high for a single molecule among many PP1c and PP2Ac binding proteins, indicating the importance of PRIP in the regulation of phosphatases relating to cellular functions. To examine whether PKA-dependent phosphorylation of PRIP also changes the binding profile (phosphatase swapping) between PP1c and PP2Ac in living cells, COS-7 cells, which contain intrinsic PP1c and PP2Ac, but no PRIP-1 are assayed. COS-7 cells transiently transfected with genes for wildtype-PRIP-1 or the T94A mutant, which fails in phosphorylation at the site relating to PP1c binding (Terunuma et al., 2004; Gao et al., 2012), were stimulated with forskolin to increase cellular cAMP causing PKA activation, followed by the analysis; The results show that the binding of PRIP-1 to PP1c is decreased by stimulation with forskolin, while that of PP2Ac to PRIP-1 is reversely increased when COS-7 cells transfected with wild-type-PRIP-1 are used. On the other hand, when COS-7 cells transfected with T94A PRIP-1 are used, such changes were not observed. We extended the physiological experiments to an animal itself; brain lysates from mice injected intraperitoneally with isoproterenol as a physiologically relevant example, and found similar binding profiles, i.e., a phosphatase swapping. Although many proteins have been identified to regulate the function of a single protein phosphatase by direct binding (Cohen, 1989), a few proteins are known to interact with multiple families of phosphatases (Takahashi et al., 1999). Such proteins often bind not only multiple phosphatases but also protein kinases to function as a scaffold to modulate the phospho-dependent signaling pathway (Pawson and Scott, 1997). We here present evidences that PRIP is a novel protein belonging to such a protein family, because we have shown that PRIP interacts with protein phosphatases, PP1c and PP2Ac, and a protein kinase, Akt (Yoshimura et al., 2001; Kanematsu et al., 2006; Fujii et al., 2010). Other examples of such multivalent adaptor proteins are integrin aIIbb3 (Gushiken et al., 2008) and CG-NAP (Takahashi et al., 1999). Of these proteins, Vijayan et al. (2004) showed that integrin aIIbb3 interacts with catalytic subunits of both PP1 and PP2A through the aIIb subunit, and only PP1, but not PP2A, dissociates by thrombin-induced aggregation or the engagement with fibrinogen in platelets. Through the binding of PP1c and PP2Ac, in addition to the binding of other enzymes, including c-src, protein tyrosine phosphatase 1B, and protein kinase Cb, integrin aIIbb3 regulates the phospho-states of downstream target proteins, thus controlling cellular functions (Vijayan et al., 2004). The binding properties of PRIP-1 to protein phosphatases shown in this article are similar to those of integrin aIIb subunit; i.e., both PRIP-1 and integrin aIIb directly interact with PP1c through consensus PP1c binding motifs and inhibit catalytic phosphatase activity. The proteins no longer bind PP1c as an active form by cellular activation to induce their own phosphorylation, but retain an increased amount of PP2Ac, resulting in accelerated de-phosphorylation of their downstream target proteins by PP1c and PP2Ac. The close proximity of the binding sites for PP1c and PP2Ac is also similar to both integrin aIIb and PRIP. The binding sites for PP1c and PP2Ac were not identical, but PP2Ac binding was inhibited by introducing the mutation into the amino acids adjacent to the PP1c consensus motif (K92A/K93N) of PRIP-1, the observation of which is also similar to that of integrin aIIb (Gushiken et al., 2008). Summarizing the results obtained so far, regulatory modes by PRIP, of protein phosphatases, PP1c and PP2Ac are depicted below (see Fig. 4). PRIP associates with the catalytic subunits of PP1 and PP2A in a mutually exclusive manner, whose catalysis is inactive and active, respectively, and recruits the phosphatases to the sites required inside cells by binding via other regions present in PRIP, including PH domain, EF hand motif, X-Y domain, C2 domain etc. When cells are stimulated for the phosphorylation of target proteins to advance the cellular signaling pathway, PRIP itself is also phosphorylated, liberating PP1c to an active form and then associating more active PP2Ac, leading to the promotion of the de-phosphorylation of target proteins to terminate the signaling pathway promptly. Thus, the presence of PRIP would generate the transient sharp phospho-regulation of target proteins; otherwise, cellular signaling proceeds improperly because of persistent phosphorylation of target proteins. Such Please cite this article in press as: Sugiyama G, et al., Phospholipase C-related but catalytically inactive protein, PRIP as a scaffolding protein for phospho-regulation, Advances in Biological Regulation (2013), http://dx.doi.org/10.1016/j.jbior.2013.07.001

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transient phospho-regulation by PRIP has already been found in PKA-dependent phospho-regulation of the b subunit of GABAA receptors (Terunuma et al., 2004; Kanematsu et al., 2006) and of SNAP-25 (Gao et al., 2012), since these substrates phosphorylated by PKA are preferentially catalyzed by PP1c. In addition, PRIP association with more active PP2Ac, in place of PP1c liberated from PRIP by the phosphorylation (phosphatase swapping), might also contribute to the regulation of events requiring protein substrates specific to PP2Ac. For instance, it was recently reported that phospho (active)-Akt in the insulin-signaling pathway is dephosphorylated by PP2Ac through complex formation along with Clk2 and B56b (Rodgers et al., 2011), so it is possible that PRIP is implicated in insulin signaling via phosphatase swapping, since PRIP binds to the phosphorylated form of Akt as reported previously (Fujii et al., 2010). Furthermore, we recently found that PP2Ac was more actively implicated in SNAP-25 phosphorylation by PKC than by PKA (Gao et al., 2012), so phosphatase swapping on PRIP might be actively implicated in PKC regulation of SNAP-25. Further works on the physiological relevance of the current findings are apparently needed. In addition, we did not include the characteristics of binding to Akt in this article, since the further experiments regarding the binding mode and the physiological relevance are being currently undertaken. We believe that the impairment of proper phospho-regulation by lacking PRIP has NO CAUSAL relationship for the malfunctions found with DKO mice described above, but missing the proper phosphoregulation mediated by PRIP might be partly underlying for explaining the phenotypes of DKO mice. Acknowledgments Our works have being supported by KAKENHI from Japan Society for Promotion of Science to MH (24229009), HT (24592805), JG (25861758) and MM (24592804), and by the Funding Program for Next Generation World-Leading Researchers from Japan Society for Promotion of Science to TK (LS087). References Ceulemans H, Bollen M. Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiological Reviews 2004;84:1–39. Cohen P. The structure and regulation of protein phosphatases. Annual Review of Biochemistry 1989;58:453–508. Doira N, Kanematsu T, Matsuda M, Takeuchi H, Nakano H, Ito Y, et al. Hyperinsulinemia in PRIP-1 gene deleted mice. Biomedical Research 2001;22:157–65. Ellis MV, James SR, Perisic O, Downes CP, Williams RL, Katan M. Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Journal of Biological Chemistry 1998;273:11650–9. Essen L-O, Perisic O, Cheung R, Katan M, Williams RL. 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