Liprin phosphorylation regulates binding to LAR: evidence for liprin autophosphorylation

The LAR transmembrane tyrosine phosphatase associates with liprin-alpha proteins and colocalizes with liprin-alpha1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extents of in vitro and in vivo phosphorylation of these mutants were reduced relative to that of wild-type liprin-alpha1. Treatment of liprin-alpha1 with calf intestinal phosphatase weakened its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibited liprin phosphorylation did not bind to LAR as assessed by coprecipitation studies. Endogenous LAR was shown to bind phosphorylated liprin-alpha1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, we found immunoprecipitates of liprin-alpha1 expressed in COS-7 cells to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-alpha1 derived from Sf-9 insect cells retained the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-alpha1 truncation mutant incorporated phosphate after denaturation and/or renaturation in SDS gels. Finally, binding assays showed that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LH region mutations that inhibit liprin phosphorylation stabilized the association of liprin with ATP-agarose. Taken together, our results suggest that liprin autophosphorylation regulates its association with LAR.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1

RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells.     

The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C

Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.     

Extracellular S100A1 protein inhibits apoptosis in ventricular cardiomyocytes via activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2)

S100A1 is a Ca2+-binding protein of the EF-hand type that belongs to the S100 protein family. It is specifically expressed in the myocardium at high levels and is considered to be an important regulator of cardiac contractility. Because the S100A1 protein is released into the extracellular space during ischemic myocardial injury, we examined the cardioprotective potential of the extracellular S100A1 protein on ventricular cardiomyocytes in vitro. In this report we show that extracellularly added S100A1 protein is endocytosed into the endosomal compartment of neonatal ventricular cardiomyocytes via a Ca2+-dependent clathrin-mediated process. S100A1 uptake protects neonatal ventricular cardiomyocytes from 2-deoxyglucose and oxidative stress-induced apoptosis in vitro. S100A1-mediated anti-apoptotic effects involve specific activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pro-survival pathway, including activation of phospholipase C, protein kinase C, mitogen-activated protein kinase kinase 1, and ERK1/2. In contrast, neither transsarcolemmal Ca2+ influx via the L-type channel nor protein kinase A activity seems to take part in the S100A1-mediated signaling pathway. In conclusion, this study provides evidence for the S100A1 protein serving as a novel cardioprotective factor in vitro. These findings warrant speculation that injury-dependent release of the S100A1 protein from cardiomyocytes may serve as an intrinsic mechanism to promote survival of the myocardium in vivo.     

Molecular mechanisms of Ca(2+) signaling in neurons induced by the S100A4 protein

The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca(2+) signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca(2+) entry via nonselective cation channels and via T- and L-type voltage-gated Ca(2+) channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders.     

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.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-alpha/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation.     

Interactions between the microtubule-associated tau proteins and S100b regulate tau phosphorylation by the Ca2+/calmodulin-dependent protein kinase II

Binding between the microtubule-associated tau protein and S100b protein was demonstrated by affinity chromatography and cross-linking experiments and was manifested in the effect of S100b on tau protein phosphorylation by protein kinase II. All three expressions of the binding showed that S100b discriminates among the four species of tau, revealing for the first time that the different kinds of tau may differ functionally. Noncovalent interaction between tau and S100b depended on the presence of Ca2+ or Zn2+ and resulted in total inhibition of tau phosphorylation by protein kinase II. In the absence of reducing agent, covalent binding studies between Cys84 beta in the carboxyl-terminal region of the S100b-beta subunit and tau proteins confirmed interactions between the two proteins. It is suggested that the homologous calcium-binding domain that characterizes the carboxyl terminus of S100 and the tubulin subunit may be responsible for the common interaction of both proteins with tau proteins. The physicochemical relationship between S100 subunits and p11, the subunit of a substrate for tyrosine kinase, and their similarity in interaction with protein kinase substrates are discussed.     

Identification of major ERK-related phosphorylation sites in Gab1

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.     

Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1

Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3beta) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G2/M phase, was enhanced by tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), and was stimulated in vitro by CDK 1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL-resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.     

Regulation of adrenergic receptor function by phosphorylation. II. Effects of agonist occupancy on phosphorylation of alpha 1- and beta 2-adrenergic receptors by protein kinase C and the cyclic AMP-dependent protein kinase

The present study was undertaken to determine the ability of protein kinase C and protein kinase A to directly phosphorylate the purified alpha 1- and beta 2-adrenergic receptors (AR). Both the catalytic subunit of protein kinase A and the protein kinase C, purified from bovine heart and pig brain, respectively, are able to phosphorylate the purified alpha 1-AR from DDT1 MF-2 smooth muscle cells. Occupancy of the receptor by an alpha 1 agonist, norepinephrine (100 microM), increases the rate of phosphorylation by protein kinase C but not by protein kinase A. The maximum stoichiometry of phosphorylation obtained is not affected by the agonist and reached 3 mol of PO4/mol of receptor for protein kinase C and 1 mol of PO4/mol of receptor for protein kinase A. The phosphopeptide maps of the trypsinized alpha 1-AR phosphorylated by each kinase differ drastically. The beta 2-AR purified from hamster lungs can also be phosphorylated by the two kinases. In contrast to the alpha 1-AR, the occupancy of the beta 2-AR by the agonist isoproterenol (20 microM) increases the rate of phosphorylation of the beta 2-AR by protein kinase A but not by protein kinase C. The maximum amount of phosphate incorporated into the receptor is not affected in either case by the agonist and reaches 1 mol of PO4/mol of receptor with protein kinase A and 0.4 mol of PO4/mol of receptor with protein kinase C. The phosphopeptide maps of the trypsinized receptor phosphorylated by either kinase reveal similar profiles. Thus, both alpha 1-AR and beta 2-AR are substrates for protein kinase A and protein kinase C. Agonist occupancy of the two receptors facilitates their phosphorylation only by the protein kinase coupled to their own signal transduction pathway. These observations suggest that "feedback" and "cross-system" phosphorylation may represent distinct and differently regulated mechanisms of modulation of receptor function.     

MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1

MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. We studied the activation of MARK and identified the upstream kinase, MARKK, a member of the Ste20 kinase family. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation.     

Angiotensin II stimulates phosphorylation of nuclear lamins via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells

Angiotensin II (ang II) induces c-fos gene expression in part via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells (VSMC). However, little is known about the mechanisms by which protein kinase C regulates nuclear functions. We examined the ability of ang II to phosphorylate nuclear lamina proteins in VSMC and the possibility that protein kinase C is involved in these putative phosphorylation events. Ang II stimulated the phosphorylation of Triton X-100- and high salt-insoluble nuclear envelope proteins with molecular weights of 70,000, 67,000, and 60,000. These proteins were identified as lamins A, B, and C, respectively, based on their mobilities on two-dimensional gel electrophoresis and interaction with antibodies to lamins as detected by immunoblot analyses. After a 2-min delay, phosphorylation levels of lamins increased, peaked at 20-30 min, and were sustained for at least 60 min after ang II stimulation. The threshold, half-maximal, and maximal concentrations of ang II which induced phosphorylation of lamins were 0.1, 0.5-1, and 100 nM, respectively. Phorbol 12-myristate 13-acetate also induced these reactions, whereas ionomycin did not. Down-regulation of protein kinase C by prolonged treatment with phorbol 12,13-dibutyrate attenuated ang II-induced phosphorylation of lamins. In vitro phosphorylation of nuclear envelope proteins by protein kinase C revealed that lamins served as substrates for this enzyme. These results indicate that ang II induces phosphorylation of lamins in cultured VSMC and suggest that protein kinase C is either directly or indirectly involved in these reactions. The results raise the possibility that phosphorylation of nuclear proteins is one of the important steps by which the protein kinase C signaling pathway regulates agonist-induced nuclear events.     

TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB

Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.