Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.     

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.     

Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.     

Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1

TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (K_a = 1.80 × 10⁶ M⁻¹) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (∼60% inhibition), mono- (∼50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.     

Phosphorylation of Merlin Regulates its Stability and Tumor Suppressive Activity

The neurofibromatosis-2 (NF2) tumor suppressor protein, merlin or schwannomin, inhibits cell proliferation by modulating the growth activities of its binding partners, including the cell surface glycoprotein CD44, membrane-cytoskeleton linker protein ezrin and PIKE (PI 3-kinase Enhancer) GTPase etc. Merlin exerts its growth suppressive activity through a folded conformation that is tightly controlled through phosphorylation by numerous protein kinases including PAK, PKA and Akt. Merlin inhibits PI 3-kinase activity through binding to PIKE-L. Now, we show that merlin is a physiological substrate of Akt, which phosphorylates merlin on both T230 and S315 residues. This phosphorylation abolishes the folded conformation of merlin and inhibits its association with PIKE-L, provoking merlin polyubiquitination and proteasome-mediated degradation. This finding demonstrates a negative feed-back loop from merlin/PIKE-L/PI 3-kinase to Akt in tumors. The proliferation repressive activity of merlin is also partially regulated by S518 phosphorylation. Thus, Akt-mediated merlin T230/S315 phosphorylation, combined with S518 phosphorylation by PAK and PKA, provides new insight into abrogating merlin function in the absence of merlin mutational inactivation.     

Cyclic AMP-dependent protein kinase phosphorylates merlin at serine 518 independently of p21-activated kinase and promotes merlin-ezrin heterodimerization

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrin-radixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKA-induced phosphorylation. In vitro [gamma-(33)P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growth-suppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.     

Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization

The Nf2 tumor suppressor gene product merlin is related to the membrane-cytoskeleton linker proteins of the band 4.1 superfamily, including ezrin, radixin, and moesin (ERMs). Merlin is regulated by phosphorylation in a Rac/cdc42-dependent fashion. We report that the phosphorylation of merlin at serine 518 is induced by the p21-activated kinase PAK2. This is demonstrated by biochemical fractionation, use of active and dominant-negative mutants of PAK2, and immunodepletion. By using wild-type and mutated forms of merlin and phospho-directed antibodies, we show that phosphorylation of merlin at serine 518 leads to dramatic protein relocalization.     

The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

Phosphorylation of Merlin Regulates its Stability and Tumor Suppressive Activity

The neurofibromatosis-2 (NF2) tumor suppressor protein, merlin or schwannomin, inhibits cell proliferation by modulating the growth activities of its binding partners, including the cell surface glycoprotein CD44, membrane-cytoskeleton linker protein ezrin and PIKE (PI 3-kinase enhancer) GTPase, etc. Merlin exerts its growth suppressive activity through a folded conformation that is tightly controlled through phosphorylation by numerous protein kinases including PAK, PKA and Akt. Merlin inhibits PI 3-kinase activity through binding to PIKE-L. Now, we show that merlin is a physiological substrate of Akt, which phosphorylates merlin on both T230 and S315 residues. This phosphorylation abolishes the folded conformation of merlin and inhibits its association with PIKE-L, provoking merlin polyubiquitination and proteasome-mediated degradation. This finding demonstrates a negative feed-back loop from merlin/PIKE-L/PI 3-kinase to Akt in tumors. The proliferation repressive activity of merlin is also partially regulated by S518 phosphorylation. Thus, Akt-mediated merlin T230/S315 phosphorylation, combined with S518 phosphorylation by PAK and PKA, provides new insight into abrogating merlin function in the absence of merlin mutational inactivation.Key Words: Akt, merlin, neurofibromatosis, phosphorylation, cell invasion and migrationNeurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by bilateral occurrence of vestibular schwannomas and other brain tumors, including meningiomas and ependymomas.¹ The NF2 tumor suppressor protein merlin belongs to the band 4.1 family of cytoskeleton-associated proteins.^2,3 Merlin isoform I possesses a “closed” conformation via an NTD (N-terminal domain)/CTD (Carboxy terminal domain) intramolecular interaction. In contrast, the alternatively spliced merlin isoform II exists in an “open” conformation that cannot function as a negative growth regulator.⁴ Merlin with NF2 patient missense mutations in the NTD or CTD exhibit an “open” conformation and do not suppress cell growth.⁵ Merlin plays a key role in regulating cell proliferation and cell migration. Merlin exerts its growth suppressive activity through intramolecular folding that dictates its binding affinities to various cellular partners including HRS (hepatocyte growth factor regulated tyrosine kinase substrate), CD44 cell surface glycoprotein, schwannomin interacting protein-1 (SCHIP1), βII-spectrin or fodrin, PIKE-L GTPase and other ERM proteins.^6–10 For instance, CD44 preferentially associates with hypophosphorylated merlin, and relatively little phosphorylated merlin binds CD44. Interference with merlin binding to CD44 impairs merlin growth suppression in RT4 rat schwannoma cells.¹¹We have previously shown that the PIKE/PI 3-kinase signaling pathway is negatively regulated by protein 4.1N, a neuronal selective isoform of band 4.1 superfamily.¹² Recently, we show that PIKE-L is an important mediator of merlin growth suppression. We show that merlin blocks cell proliferation by inhibiting PI 3-kinase through binding to PIKE-L. Interestingly, wild-type merlin, but not patient-derived mutant (L64P), binds PIKE-L and inhibits PI 3-kinase activity. This suppression of PI 3-kinase activity results from merlin disrupting the binding of PIKE-L to PI 3-kinase. Mutation of PIKE-L with Proline 187 into Leucine disrupts its interaction with merlin. Accordingly, merlin suppression of PI 3-kinase activity as well as schwannoma cell growth is abrogated by a single PIKE-L point mutation (P187L).¹⁰Merlin is phosphorylated on S518 by members of the PAK family of kinases, including PAK1 and PAK2,^13–15 which mislocates merlin from the plasma membrane to the cytoplasm. A merlin mutant that mimics S518 phosphorylation (S518D) cannot suppress cell growth or motility in RT4 rat schwannoma cells, and leads to dramatic changes in cell morphology and actin cytoskeleton organization.¹⁶ S518 phosphorylation results in impaired merlin NTD/CTD folding as well as altered interactions with critical merlin associated proteins, including CD44 and HRS.¹⁷ Recently, Alfthan and colleagues demonstrated that Protein Kinase-A (PKA) induces merlin phosphorylation on both N-and C-terminal residues.¹⁸ In addition to S518 phosphorylation, PKA can phosphorylate merlin at S66 in the N-terminal domain (Fig. 1). When PAK activity is suppressed, merlin can still be phosphorylated by PKA in cells, indicating that these two kinases function independently. The N-terminus of ezrin strongly binds to a PKA-phosphorylated, but not unphosphorylated, merlin CTD. In contrast, PAK2-induced S518 phosphorylation has a minimal effect on the interaction between full-length merlin and full-length ezrin.¹⁷ Besides regulation of cell growth, merlin also mediates cell motility presumably through directly binding to actin cytoskeleton.¹⁹ Depletion of merlin in normal fibroblast results in enhanced cell invasion. Nevertheless, expression of merlin attenuates Y397 phosphorylation on FAK, an essential player in cell migration and invasion. This observation might provide a molecular mechanism accounting for merlin inhibitory activity in cell motility.²⁰Open in a separate windowFigure 1Merlin phosphorylation sites by various kinases.In addition to PAK and PKA, we show that Akt potently phosphorylates merlin at both T230 and S315 residues. Blocking one site phosphorylation abolishes the other site phosphorylation by Akt, indicating that these two phosphorylation sites are mutually regulated.²¹ The physiological significance of the tight control on merlin phosphorylation by Akt remains incompletely understood. Presumably, only when mitogenic signal or oncogenic stress is strongly enough to provoke cell proliferation or migration, does Akt simultaneously phosphorylate both sites. Akt phosphorylation of merlin attenuates the NTD/CTD interaction and inhibits its binding activity to PIKE-L, CD44 and ezrin. Further, phosphorylation mediates the biological activities of merlin, as expression of a phosphomimetic merlin mutant (T230DS315D) increases cell motility and proliferation in a rat schwannoma cell line (Fig. 2). By contrast, expression of a mutant (T230A/S315A) that was unable to undergo phosphorylation inhibited cell growth and motility. The F1 motif in FERM proteins including merlin exhibits an ubiquitin-like structure. This domain facilitates MDM2 degradation and stimulates the ubiquitination and degradation of TRBP, a double-stranded RNA binding protein. Surprisingly, inhibition of the proteasome does not affect total merlin protein levels in human glioblastoma cells, but leads to a marked increase of phospho-S315 merlin. Simultaneous treatment with MG132, which blocks proteasome-mediated degradation and PI 3-kinase inhibitor, wortmannin, which inhibits Akt phosphorylation of merlin, substantially enhances merlin levels. Coimmunoprecipitation studies demonstrate that Akt-phosphorylated merlin is rapidly ubiquitinated, presumably by spectrin, which binds to merlin and possesses ubiquitin-conjugating and ubiquitin E3 ligase function. However, S518 phosphorylation fails to trigger merlin ubiquitination, suggesting that Akt, but not PAK or PKA, phosphorylation selectively elicits merlin ubiquitination. Using a panel of human primary nervous system tumors, we found that merlin phosphorylation by Akt also mediates its degradation in primary tumors. Accordingly, tumors that possess high levels of phospho-Akt exhibited low levels of merlin. Therefore, our data suggest a novel role for Akt in promoting phosphorylation and subsequent degradation of merlin. Loss of merlin has been linked to schwannomas and other nervous system tumors, and these results indicate that inhibitors for PI 3-kinase/Akt pathway might restore merlin function in tumors.Open in a separate windowFigure 2The model for Akt interaction with merlin and its phosphorylation.     

Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50

Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCalpha and beta isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.     

TIMAP,a novel CAAX box protein regulated by TGF-beta1 and expressed in endothelial cells

Representational difference analysis ofthe glomerular endothelial cell response to transforming growthfactor-1 (TGF-1) revealed a novel gene, TIMAP (TGF--inhibitedmembrane-associated protein), which contains 10 exons and maps to humanchromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressedin all cultured endothelial and hematopoietic cells. The frequency ofthe TIMAP SAGE tag is much greater in endothelial cell SAGE databasesthan in nonendothelial cells. Immunofluorescence studies of rat tissuesshow that anti-TIMAP antibodies localize to vascular endothelium.TGF-1 represses TIMAP through a protein synthesis- and histonedeacetylase-dependent process. The TIMAP protein contains five ankyrinrepeats, a protein phosphatase-1 (PP1)-interacting domain, aCOOH-terminal CAAX box, a domain arrangement similar to that of MYPT3,and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion proteinlocalized to the plasma membrane in a CAAX box-dependent fashion.Hence, TIMAP is a novel gene highly expressed in endothelial andhematopoietic cells and regulated by TGF-1. On the basis of itsdomain structure, TIMAP may serve a signaling function, potentiallythrough interaction with PP1.

     

Deactivation of sphingosine kinase 1 by protein phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.     

The protein phosphatase-1 targeting subunit TIMAP regulates LAMR1 phosphorylation

TIMAP is a prenylated endothelial cell protein with a domain structure that predicts it to be a protein phosphatase-1 (PP-1) regulatory subunit. We found that TIMAP interacts with the 37/67 kDa laminin receptor (LAMR1) in yeast two-hybrid assays. In endothelial cells, endogenous TIMAP and LAMR1 co-immunoprecipitated and co-localized at the plasma membrane. TIMAP amino acids 261-290, representing the fourth ankyrin repeat of TIMAP, are necessary and sufficient for the interaction. In MDCK cells, lacking endogenous TIMAP, overexpression of full-length TIMAP, but not TIMAP deleted in the fourth ankyrin domain, allowed co-immunoprecipitation with LAMR1. PP-1 co-precipitated with overexpressed and endogenous TIMAP in MDCK and endothelial cells, respectively. In MDCK cells, PP-1 associated with LAMR1 in the presence, but not in the absence, of TIMAP. LAMR1 was a substrate for PP-1 in vitro, and in MDCK cells its phosphorylation was abrogated by expression of full-length TIMAP but not by TIMAP deficient in the fourth ankyrin domain. Hence, TIMAP targets PP-1 to LAMR1, and LAMR1 is a TIMAP-dependent PP-1 substrate.     

A critical role for the protein phosphatase 2A B'α regulatory subunit in dephosphorylation of sphingosine kinase 1

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.     

Activation of p21-activated kinase 2 by human immunodeficiency virus type 1 Nef induces merlin phosphorylation

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.     

Relationship between ATM and Ribosomal Protein S6 Revealed by the Chemical Inhibition of Ser/Thr Protein Phosphatase Type 1

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.     

TIMAP is a positive regulator of pulmonary endothelial barrier function

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.     

EBP50 phosphorylation by Cdc2/Cyclin B kinase affects actin cytoskeleton reorganization and regulates functions of human breast cancer cell line MDA-MB-231

The actin cytoskeleton plays an important role in cell shape determination, adhesion and cell cycle progression. Ezrinradixin-moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na⁺-H⁺ exchanger regulatory factor 1 (NHERF1), associates with actin cytoskeleton and is related to cell cycle progression. Its Ser279 and Ser301 residues are phosphorylated by cyclin-dependent kinase 2 (cdc2)/cyclin B during the mitosis phase. However, the biological significance of EBP50 phosphorylation mediated by cdc2/cyclin B is not clear. In the present study, MDA-MB-231 cells with low levels of endogenous EBP50 protein were stably transfected with constructs of EBP50 wild type (WT), phosphodeficient (serine 279 and serine 301 mutated to alanine-S279A/S301A) or phospho-mimetic (serine 279 and serine 301 mutated to aspartic acid-S279D/S301D) mutants. Subsequently, multiple phenotypes of these cells were characterized. Failure of cdc2/cyclin B-mediated EBP50 phosphorylation in cells expressing S279A/S301A (AA cells) significantly increased F-actin content, enhanced the adherence of cells to the extracellular matrix, altered cell morphology and caused defects in cytokinesis, as reflected in the formation of giant cells with heteroploid DNA and multinucleation or giant nuclei. Furthermore, knockdown of EBP50 expression in AA cells rescued cell defects such as the cytokinesis failure and abnormal cell morphology. EBP50 S279A/ S301A had a weaker binding affinity with actin than EBP50 S279D/S301D, which might explain the increase of F-actin content in the AA cells. The present results suggest that cdc2/cyclin B-mediated EBP50 phosphorylation may play a role in the regulation of various cell functions by affecting actin cytoskeleton reorganization.     

Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin''s ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70^MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70^MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70^MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70^MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.