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Emerging evidence suggests critical roles for protein phosphatase 2A (PP2A) in islet beta cell function, including survival and demise (Kowluru A: Biochemical Pharmacol 69:1681-1691, 2005). Herein, we identified an okadaic acid (OKA)-sensitive PP2A-like phosphatase in the nuclear fraction from insulin-secreting INS-1 cells. Western blot analysis indicated relatively higher abundance of the catalytic subunit of protein phosphatase 4 (PP4c) compared to PP2Ac in this fraction. Autoradiographic and vapor-phase equilibration analyses suggested that the nuclear PP4c undergoes OKA-sensitive carboxylmethylation (CML) when S-adenosyl-L-((3)H-methyl) methionine (SAM) was used as the methyl donor. Exposure of INS cells to interleukin-1beta (IL-1beta; 600 pM; 48 h) resulted in a marked increase in nitric oxide (NO) release with concomitant reduction in the degree of expression, the CML and the catalytic activity of only PP4, but not PP2A, in the nuclear fraction. Immunoprecipitation studies suggested potential complexation of PP4c with nuclear lamin-B, a key regulatory protein involved in the nuclear envelope assembly. Based on these findings, we propose that IL-1beta-mediated inhibition of PP4 activity might result in the retention of lamin-B in its phosphorylated state, which is a requisite for its degradation by caspases leading to the apoptotic demise of the beta cell (Veluthakal et al.: Am J Physiol Cell Physiol 287:C1152-C1162, 2004).
A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B', B'' gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aalpha scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aalpha paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aalpha knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aalpha with a mutant with selective B' subunit binding deficiency implicated PP2A/B' heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Balpha and Bdelta led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Balpha and Bdelta in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties.
Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.
Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.
BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on post-translational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events - mediated through separate signaling processes and involving different target sites - in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERK-inducible versus ERK-independent sites.
Butyric acid is well recognized as a histone deacetylase (HDAC) inhibitor, and changes in histone acetylation are thought to alter gene expression. The mechanism by which sodium butyrate (NaB) induces p21WAF1/CIP1, a critical gene involved in the antiproliferative effect of NaB, was studied at the chromatin level. Using chromatin immunoprecipitation (ChIP) assay, acetylation of histone H3 was observed at the proximal region of the promoter within 30 min of NaB exposure and this extended to the distal region within 2 hr. By contrast, histone H4 was acetylated both at the proximal and the distal regions of the promoter within 30 min. NaB did not influence other histone modifications. NaB stimulated recruitment of the transcription factors ZBP89 and Sp1 as well as GCN5, but did not influence recruitment of Sp3, HDAC1, p300, or CBP. As recruitment of HDAC1 to the promoter appeared not to account for NaB-induced changes in histone acetylation, we aimed to influence HDAC activity by altering its phosphorylation status. The kinase inhibitor, H7, suppressed p21WAF1/CIP1 mRNA in both the absence and the presence of NaB without influencing the butyrate-induced hyperacetylation of H3 and H4 associated with the p21WAF1/CIP1 promoter. These results suggest that acetylation of histones at the p21WAF1/CIP1 promoter is not sufficient for NaB to exert antiproliferative effects via transcription of the p21WAF1/CIP1 gene. Induction of p21WAF1/CIP1 transcription by the phosphatase inhibitor, okadaic acid, in the absence of changes in association of acetylated histones with the p21WAF1/CIP1 promoter provides further evidence of the importance of phosphorylation to p21WAF1/CIP1 transcription.
Copyright 2003 Wiley-Liss, Inc.
The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.
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.
BAD is a proapoptotic member of the BCL-2 family of proteins, which play a major role in regulating apoptosis in cytokine-dependent hematopoietic cells. The function of BAD is regulated by reversible phosphorylation. Deprivation of survival factors induces BAD dephosphorylation, resulting in apoptosis. Serine-threonine phosphatase activity dephosphorylated BAD in interleukin-3-dependent FL5.12 lymphoid cells. Inhibition of PP2A activity by treatment of cells with PP2A-selective inhibitors, okadaic acid and fostriecin, prevented BAD dephosphorylation in these cells. Conversely, BAD dephosphorylation was not inhibited by the PP1-selective inhibitor tautomycin. In cell-free extracts, BAD phosphatase activity was also inhibited by the PP2A-selective inhibitors okadaic acid and fostriecin, but not by the PP1-specific protein inhibitor I-2. Dissociation of 14-3-3 from BAD was a prerequisite for BAD dephosphorylation in vitro, suggesting a mechanism by which 14-3-3 can regulate the activation of the proapoptotic function of BAD in vivo. Significantly, the inhibition of BAD phosphatase activity rescued cell death induced by survival factor withdrawal in FL5.12 cells expressing wild-type BAD but not phosphorylation-defective mutant BAD. These data indicate that PP2A, or a PP2A-like enzyme, dephosphorylates BAD and, in conjunction with 14-3-3, modulates cytokine-mediated survival.
Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.