Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

Phosphorylation-independent association of CXCR2 with the protein phosphatase 2A core enzyme

Protein phosphatase 2A (PP2A) is postulated to be involved in the dephosphorylation of G protein-coupled receptors. In the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac and PR65, but not with the PP2Ac monomer, suggesting direct interaction of the receptor with PR65. The integrity of a sequence motif in the C terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC chemokine receptors, is required for the receptor binding to the PP2A core enzyme. CXCR2 co-immunoprecipitates with the PP2A core enzyme in HEK293 cells and in human neutrophils. Overexpression of dominant negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks the receptor association with the PP2A core enzyme, and an internalization-deficient mutant form of CXCR2 (I323A,L324A) also exhibits impaired association with the PP2A core enzyme, suggesting that the receptor internalization is required for the receptor binding to PP2A. A phosphorylation-deficient mutant of CXCR2 (331T), which has previously been shown to undergo internalization in HEK293 cells, binds to an almost equal amount of the PP2A core enzyme in comparison with the wild-type CXCR2, suggesting that the interaction of the receptor with PP2A is phosphorylation-independent. The dephosphorylation of CXCR2 is reversed by treatment of the cells with okadaic acid. Moreover, pretreatment of the cells with okadaic acid increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated calcium mobilization and chemotaxis. Taken together, these data indicate that PP2A is involved in the dephosphorylation of CXCR2. We postulate that this interaction results from direct binding of the regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2 after receptor sequestration and internalization.     

The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated β-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.     

Integrin cross-talk in endothelial cells is regulated by protein kinase A and protein phosphatase 1

In endothelial cells (ECs) beta1 integrin function-blocking antibodies inhibit alphavbeta3 integrin-mediated adhesion to a recombinant alpha4-laminin fragment (ralpha4LN fragment). beta1 integrin sequestration of talin is not the mechanism by which beta1 integrin modulates alphavbeta3 integrin ligand binding. Rather, treatment of the ECs with beta1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta1 integrin function-blocking antibodies on beta3 integrin serine phosphorylation and EC-ralpha4LN fragment binding. beta3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alphavbeta3 integrin-ralpha4LN fragment binding, we mutated it to alanine (beta3S752A) or aspartic acid (beta3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta3S752A integrin attach robustly to ligand. CHO cells expressing beta3S752D integrin do not. Because the beta3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta3 integrin. Instead, we have tested an hypothesis that PKA regulates beta3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alphavbeta3-mediated cell adhesion to the ralpha4LN fragment when both PKA and beta1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta1 integrin negatively modulates alphavbeta3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.     

Cell cycle dependent exposure of a high molecular weight protein on the surface of mouse L cells

The non-penetrating lactoperoxidase iodination probe has been employed in conjunction with synchronously dividing populations of mouse L cells to identify a high molecular weight protein which is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of this structure, called band 1, to lactoperoxidase-catalyzed iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of L cell growth.     

Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae.

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G(2)/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G(1)/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

Cell cycle dependent variation of calmodulin in Tetrahymena

The level of calmodulin (CaM), a ubiquitous calcium-binding protein of eukaryotic cells was determined at different phases of the cell cycle in a synchronized Tetrahymena population. It was found that the concentration of CaM at G1 was approximately half of the concentration of S and this 2 x G1 level of CaM was maintained through the G2 and M stages of the cell cycle. To ascertain the role of CaM in the initiation of DNA synthesis, the cells were treated with trifluoperazine (TFP), a CaM antagonist, and EGTA (Ca2+-chelator) at the G1/S boundary. It was found that DNA synthesis was inhibited in these drug-treated cells. The uptake of the nucleotide precursor was not affected in TFP and EGTA treated cells, thus excluding the possibility of alteration in the membrane transport properties. Treatment with TFP failed to inhibit the synchronous mitotic division in Tetrahymena. The existence of a variable content of CaM through the cell cycle of Tetrahymena was demonstrated, suggesting the possible involvement of this Ca2+-binding protein in the nuclear DNA replication process.     

Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2

Exposure to hypoxia causes structural changes in the endothelial cell layer that alter its permeability and its interaction with leukocytes and platelets. One of the well characterized cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers. This report describes cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process. The hypoxia-induced actin redistribution appears to be mediated by components downstream of MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia. Our results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of one of its substrates, HSP27. Because HSP27 phosphorylation is known to alter actin distribution in response to other stimuli, we postulate that it also causes the actin redistribution observed in hypoxia. This notion is supported by the observations that similar actin redistribution occurs in cells overexpressing constitutively active MK2 or phosphomimicking HSP27 mutant. Overexpressing dominant negative MK2 blocks the effects of hypoxia on the actin cytoskeleton. Taken together these results indicate that hypoxia stimulates the p38-MK2-HSP27 pathway leading to significant alteration in the actin cytoskeleton.     

I1PP2A affects tau phosphorylation via association with the catalytic subunit of protein phosphatase 2A

In Alzheimer disease (AD) brain, the level of I (1)(PP2A), a 249-amino acid long endogenous inhibitor of protein phosphatase 2A (PP2A), is increased, the activity of the phosphatase is decreased, and the microtubule-associated protein Tau is abnormally hyperphosphorylated. However, little is known about the detailed regulatory mechanism by which PP2A activity is inhibited by I (1)(PP2A) and the consequent events in mammalian cells. In this study, we found that both I (1)(PP2A) and its N-terminal half I (1)(PP2A(1-120)), but neither I (1)(PP2A(1-163)) nor I (1)(PP2A(164-249)), inhibited PP2A activity in vitro, suggesting an autoinhibition by amino acid residues 121-163 and its neutralization by the C-terminal region. Furthermore, transfection of NIH3T3 cells produced a dose-dependent inhibition of PP2A activity by I (1)(PP2A)(1). I (PP2A) and PP2A were found to colocalize in PC12 cells. I (1)(PP2A) could only interact with the catalytic subunit of PP2A (PP2Ac) and had no interaction with the regulatory subunits of PP2A (PP2A-A or PP2A-B) using a glutathione S-transferase-pulldown assay. The interaction was further confirmed by coimmunoprecipitation of I (1)(PP2A) and PP2Ac from lysates of transiently transfected NIH3T3 cells. The N-terminal isotype specific region of I (1)(PP2A) was required for its association with PP2Ac as well as PP2A inhibition. In addition, the phosphorylation of Tau was significantly increased in PC12/Tau441 cells transiently transfected with full-length I (1)(PP2A) and with PP2Ac-interacting I (1)(PP2A) deletion mutant 1-120 (I (1)(PP2A)DeltaC2). Double immunofluorescence staining showed that I (1)(PP2A) and I (1)(PP2A)DeltaC2 increased Tau phosphorylation and impaired the microtubule network and neurite outgrowth in PC12 cells treated with nerve growth factor.     

Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit.     

Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

Cell cycle dependent resistance to staphylococcal delta toxin-induced lysis of cultured cells

Staphylococcal delta toxin is a protein capable of rapidly disrupting cell membranes. Synchronized populations of 3T3 mouse fibroblasts in mitosis and early G₁ phases of the cell cycle exhibit resistance to delta toxin at concentrations cytolytic to interphase cells. Similar results were obtained with HeLa cells grown attached or in suspension culture. Increased resistance appears to result from structural or biochemical features other than cell rounding or detachment. Delta toxin stimulated significantly less cellular phospholipase A₂ (a potentially lytic enzyme activity) in mitotic 3T3 cells than in interphase cells.     

Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells

The expression and stability of the proliferation-associated nuclear antigen detected by Ki-67 antibody have been investigated in human promyelocytic leukaemic HL-60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki-67 antigen during S exceeded the increase in DNA content, and thus the Ki-67/DNA ratio rose 80% from late G1 to G2 + M. This antigen rapidly disappeared from post-mitotic cells. The half-life of this protein estimated in post-mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6-8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 microgram/ml cycloheximide, the half-life of the Ki-67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase-specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki-67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non-cycling cells.     

Cell cycle dependent exposure of a high molecular weight protein on the surface of mouse L cells.

The non-penetrating lactoperoxidase iodination probe has been employed in conjunction with synchronously dividing populations of mouse L cells to identify a high molecular weight protein is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in tha availability of this structure, called band 1, to lactoperoxidase-catalyzed iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of L cell growth.     

Yes-associated protein 65 localizes p62(c-Yes) to the apical compartment of airway epithelia by association with EBP50

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.     

Cell cycle dependent genes inducible by different mitogens in cells from different species

Summary A number of genes and cDNA sequences (including at least four oncogenes) are known to be expressed in a cell cycle-dependent manner, i.e. the levels of specific mRNAs vary with the phases of the cell cycle. In order to explore the significance of some of these sequences in the mitogenic response, we have investigated the expression of 8 cell cycle-dependent sequences (plus two control sequences, not expressed in a cell cycle-dependent manner) under a variety of conditions. These conditions included cells of different types, from different species, stimulated to proliferate by different mitogens. The genes (or sequences) studied included five cDNA clones whose sequences are preferentially expressed in early G₁, i.e. two cDNA clones inducible by platelet-derived growth factor (JE-3 and KC-1), and three cDNA clones inducible by serum (2A9, 2F1, 4F1); and three oncogenes (c-myc, c-ras^Ha and p53) whose expression is known to be cell cycle-dependent. All of the tested genes, except 2A9, c-ras^Ha and the control genes, are expressed in a cell cycle-dependent manner in human peripheral blood mononuclear cells stimulated by phytohemagglutinin and in serum-stimulated mouse and Syrian hamster fibroblasts. The inducibility of these genes by different mitogens in cells of different types and from different species strongly suggests that these genes play a role in cell cycle progression. This conclusion is further supported by the known structural and functional similarities between cell-cycle dependent genes, oncogenes and genes coding for cell-cycle related molecules.