Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Characterization of the phosphorylation status of the hepatitis B virus X-associated protein 2

The cytosolic Ah receptor (AhR) heterocomplex consists of one molecule of the AhR, a 90-kDa heat shock protein (Hsp90) dimer, and one molecule of the hepatitis B virus X-associated protein 2 (XAP2). Serine residues 43,53,131-2, and 329 on XAP2-FLAG were identified as putative phosphorylation sites using site-directed mutagenesis followed by two-dimensional phosphopeptide mapping analysis. Protein kinase CK2 (CK2) was identified as the 45-kDa kinase from COS 1 cell or liver extracts that was responsible for phosphorylation of serine 43 in the XAP2 peptide 39-57. Loss of phosphorylation at any or all of the serine residues did not significantly affect the ability of XAP2-FLAG to bind to the murine AhR in rabbit reticulocyte lysate or Hsp90 in COS-1 cells. Furthermore, all of these serine mutants were able to sequester murine AhR-YFP into the cytoplasm as well as wild-type XAP2. YFP-XAP2 S53A was unable to enter the nucleus, indicating a potential role of phosphorylation in nuclear translocation of XAP2.     

Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.     

Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity

The activity of NF-kappaB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-kappaB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.     

14-3-3beta is a p90 ribosomal S6 kinase (RSK) isoform 1-binding protein that negatively regulates RSK kinase activity

p90 ribosomal S6 kinase 1 (RSK1) is a serine/threonine kinase that is activated by extracellular signal-related kinases 1/2 and phosphoinositide-dependent protein kinase 1 upon mitogen stimulation. Under basal conditions, RSK1 is located in the cytosol and upon stimulation, RSK1 translocates to the plasma membrane where it is fully activated. The ability of RSK1 to bind the adapter protein 14-3-3beta was investigated because RSK1 contains several putative 14-3-3-binding motifs. We demonstrate that RSK1 specifically and directly binds 14-3-3beta. This interaction was dependent on phosphorylation of serine 154 within the motif RLSKEV of RSK1. Binding of RSK1 to 14-3-3beta was maximal under basal conditions and decreased significantly upon mitogen stimulation. After 5 min of serum stimulation, a portion of 14-3-3beta and RSK1 translocated to the membrane fraction, and immunofluorescence studies demonstrated colocalization of RSK1 and 14-3-3beta at the plasma membrane in vivo. Incubation of recombinant RSK1 with 14-3-3beta decreased RSK1 kinase activity by approximately 50%. Mutation of RSK1 serine 154 increased both basal and serum-stimulated RSK activity. In addition, the epidermal growth factor response of RSK1S154A was enhanced compared with wild type RSK. The amount of RSK1S154A was significantly increased in the membrane fraction under basal conditions. Increased phosphorylation of two sites essential for RSK1 kinase activity (Ser(380) and Ser(363)) in RSK1S154A compared with RSK1 wild type, demonstrated that 14-3-3 interferes with RSK1 phosphorylation. These data suggest that 14-3-3beta binding negatively regulates RSK1 activity to maintain signal specificity and that association/dissociation of the 14-3-3beta-RSK1 complex is likely to be important for mitogen-mediated RSK1 activation.     

A single serine residue at position 375 of VP16 is critical for complex assembly with Oct-1 and HCF and is a target of phosphorylation by casein kinase II.

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C-terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C-terminal boundary for complex formation with Oct-1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild-type and mutant proteins, we show that it is the main CKII site in the body of the N-terminal complex-forming region. This site is also phosphorylated in nuclear extracts. Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virtually eliminates complex formation. This serine lies in a surface-exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.     

Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.     

Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 <Emphasis Type="Italic">in vitro</Emphasis>

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.     

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.     

HSP27 regulates IL-1 stimulated IKK activation through interacting with TRAF6 and affecting its ubiquitination

Heat shock protein 27 (HSP27) is an ubiquitiously expressed protein, which has been mediated in various biological functions. Here, we present a novel mechanism utilized by HSP27 in regulating IL-1beta induced NF-small ka, CyrillicB activation. Both over-expression and RNAi experiments indicate that HSP27 physically interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and promotes TRAF6 ubiquitination. Over-expressed HSP27 augments IL-1beta induced TRAF6 ubiquitination and Ismall ka, CyrillicB kinase (IKK) activation. On the other hand, IL-1beta stimulation reduces endogenous HSP27/TRAF6 association, but inhibiting HSP27 phosphorylation by using SB202190, an inhibitor of p38, and MAPKAPK2 RNAi increases HSP27/TRAF6 association and thereby enhances TRAF6 ubiquitination, IKK phosphorylation as well as NF-small ka, CyrillicB activation. Furthermore, co-transfection study shows that HSP27 S78/82A, two phosphorylated serine site deficient mutants, but not wild-type HSP27 (HSP27 WT) and HSP27 S15A mutant increases TRAF6 ubiquitination and thereby mediates IL-1beta triggered IKK phosphorylation. Taken together, our data indicate that HSP27 regulates IL-1beta triggered NF-small ka, CyrillicB activation via a feedback loop which includes the interaction between HSP27 phosphorylation and ability of HSP27 to bind with TRAF6. The findings of this study reveal a novel mechanism by which HSP27 controls cytokine stimulation.     

Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.