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...
PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.
The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into PI(3,4,5)P3 and the subsequent PI(3,4,5)P3 signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P2-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P2 binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P3 production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P2 was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K, and the suppressor activity of PRIP in PI(4,5)P2 metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.
Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in thermoneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferatoractivated receptor-␥ coactivator-1␣). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.Obesity, which develops due to chronic excess food intake that exceeds total energy expenditure, is becoming an epidemic worldwide. Obesity is a risk factor for many chronic diseases, such as type 2 diabetes mellitus, cardiovascular disease, dyslipidemia, obstructive sleep apnea, and certain forms of cancer, which is decreasing both the quality and length of life, and increasing individual and national healthcare costs (1). To maintain energy homeostasis at the appropriate level for a given environmental condition, a complex interplay exists between the central nervous system and the peripheral organs. In the periphery, nutrient levels are regulated in key storage organs (e.g. fat in adipose tissue and glycogen in the liver and elsewhere) as well as in the blood (e.g. blood glucose) (2).In mammals, adipose tissue exists as mainly as two different types: white adipose tissue (WAT) 3 and brown adipose tissue (BAT). As the major form of energy storage, fat in white adipocytes provides a buffer for energy imbalances when energy intake is not equal to energy output; i.e. excessive energy is stored as triglyceride (TAG) and is supplied to the body by lipolysis in a nutrient-starved state. Brown adipocytes directly dissipate the chemical energy in fatty acids through uncoupling protein 1 (UCP1); i.e. the enzyme uncouples respiration from ATP synthesis and dissipates the energy as heat (3).Recently, many studies have shown that BAT participates in adult human obesity, and activation of UCP1-mediated thermogenesis in BAT prevents obesity and diabetes (3-5). Therefore, BAT has attracted much attention as a target for the treat...
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