C-terminal Phosphorylation Sites Research Articles

Human beta(3)-adrenoreceptors couple to KvLQT1/MinK potassium channels in Xenopus oocytes via protein kinase C phosphorylation of the KvLQT1 protein.

Modulation of the slow component of the delayed rectifier potassium current (IKs) in heart critically affects cardiac arrhythmogenesis. Its current amplitude is regulated by the sympathetic nervous system. However, the signal transduction from the beta-adrenergic system to the KvLQT1/MinK (KCNQ1/KCNE1) potassium channel, which is the molecular correlate of the IKs current in human cardiomyocytes, is not sufficiently understood. In the human heart, three subtypes of beta-adrenergic receptors (beta(1-3)-ARs) have been identified. Only beta(1)- and beta(3)-ARs have been shown so far to be involved in the regulation of IKs. Special interest has been paid to the regulation of IKs by the beta(3)-AR because of its potential importance in congestive heart failure. In heart failure beta(1)-ARs are known to be down regulated while the density of beta(3)-ARs is increased. Unfortunately, studies on the modulation of IKs by beta(3)-AR revealed conflicting results. We investigated the functional role of protein kinase C (PKC) in the signal transduction cascade between beta3-adrenergic receptors and IKs by expressing heterologously its molecular components, the KvLQT1/MinK potassium channel, together with human beta(3)-AR in Xenopus oocytes. Membrane currents were measured with the double electrode voltage-clamp technique. Using activators and inhibitors of PKC we demonstrated that PKC is involved in this regulatory process. Experiments in which the putative C-terminal PKC-phosphorylation sites in the KvLQT1 protein were destroyed by site directed mutagenesis reduced the isoproterenol-induced current to 27+/-3,5% compared to control. These results indicate that the amplitude of KvLQT1/MinK current is mainly increased by PKC activation. Our results suggest that the regulation of the KvLQT1/MinK potassium channel via beta(3)-AR is substantially mediated by PKC phosphorylation of the KvLQT1 protein at its four C-terminal PKC phosphorylation sites.

Cold-inducible expression of the cell division cycle gene CDC48 and its promotion of cell proliferation during cold acclimation in zebrafish cells

A member of the ATPases associated with diverse cellular activities (AAA) family, the cell division cycle gene CDC48/VCP (valosin-containing protein)/p97, was cloned from zebrafish and found to be a major cold-inducible protein in fish cells. CDC48 mRNA levels increased significantly after reducing the temperature from 30 to 15°C for 25 days. CDC48 protein levels also increased 2.5-fold after 30 days at cold temperatures. When fish cells overexpressing CDC48 were exposed to a temperature of 15°C, cell proliferation was markedly enhanced in comparison with control cells. By contrast, expression of a mutant molecule with a tyrosine-805 to alanine substitution at the C-terminal phosphorylation site inhibited cell proliferation and induced apoptosis at low temperatures. Therefore, CDC48 may promote cell cycling and cell proliferation via C-terminal tyrosine phosphorylation during cold acclimation in fish cells.

Signal-induced ubiquitination of p57 Kip2 is independent of the C-terminal consensus Cdk phosphorylation site

The cyclin-dependent kinase inhibitor p57 Kip2 is required for normal mouse embryonic development. p57 Kip2 consists of four structurally distinct domains in which the conserved C-terminal nuclear targeting domain contains a putative Cdk phosphorylation site (Thr 342) that shares a great similitude in the adjacent sequences with p27 Kip1 but not with p21 Cip1. Phosphorylation on Thr 187 has been shown to promote degradation of p27 Kip1. Although there is sequence homology between the C-terminal part of p27 Kip1 and p57 Kip2, we show that the ubiquitination and degradation of p57 Kip2 are independent of Thr 342. In contrast a destabilizing element located in the N-terminal is implicated in p57 Kip2 destabilization.

Phosphorylation of the NADPH oxidase component p67(PHOX) by ERK2 and P38MAPK: selectivity of phosphorylated sites and existence of an intramolecular regulatory domain in the tetratricopeptide-rich region.

p67(PHOX), a cytosolic component of the NADPH oxidase complex, is phosphorylated during neutrophil activation by several agonists. The intracellular signaling pathways leading to its phosphorylation in neutrophils may involve a PKC-dependent pathway and a PKC-independent pathway. Here, we analyzed p67(PHOX) phosphorylation by ERK2 and p38MAPK. Both ERK2 and p38MAPK phosphorylated p67(PHOX) in vitro, with similar K(m) values (10 and 9 microM, respectively). Phosphopeptide mapping indicated that ERK2 and p38MAPK phosphorylate different subgroups of peptides. Using truncated forms of p67(PHOX), we found that the major phosphorylation target site of ERK2 was located in the N-terminal fragment (1-243), while the major phosphorylation target sites of p38MAPK were located in the C-terminal fragment (244-526). Furthermore, an additional peptide, which was not phosphorylated in the intact protein, appeared to be phosphorylated in the isolated C-terminal fragment (aa 244-526). This site may not thus be accessible in the intact protein. Indeed, incubation of the C-terminal fragment (244-526) with different N-terminal fragments (1-243, 1-210, or 1-199) containing the tetratricopeptide-rich region prevented phosphorylation of this C-terminal fragment. ERK1/2 and p38MAPK are also involved in p67(PHOX) phosphorylation in intact neutrophils. Indeed, PD98059 and SB203580, two selective inhibitors of MEK1/2 and p38MAPK, respectively, inhibited p67(PHOX) phosphorylation in fMLP- and PMA-stimulated neutrophils, with additive effects, thus suggesting that they also target different sites in vivo. Furthermore, the major peptides phosphorylated by ERK2 and p38MAPK in vitro were also phosphorylated in fMLP-stimulated neutrophils. Taken together, these results suggest not only that p67(PHOX) is phosphorylated by ERK2 and p38MAPK in vitro and in intact neutrophils on several selective sites but also that a C-terminal phosphorylation site may become accessible after a conformational change of the protein.

Modulation of the Kv3.1b Potassium Channel Isoform Adjusts the Fidelity of the Firing Pattern of Auditory Neurons

Neurons of the medial nucleus of the trapezoid body, which transmit auditory information that is used to compute the location of sounds in space, are capable of firing at high frequencies with great temporal precision. We found that elimination of the Kv3.1 gene in mice results in the loss of a high-threshold component of potassium current and failure of the neurons to follow high-frequency stimulation. A partial decrease in Kv3.1 current can be produced in wild-type neurons of the medial nucleus of the trapezoid body by activation of protein kinase C. Paradoxically, activation of protein kinase C increases temporal fidelity and the number of action potentials that are evoked by intermediate frequencies of stimulation. Computer simulations confirm that a partial decrease in Kv3.1 current is sufficient to increase the accuracy of response at intermediate frequencies while impairing responses at high frequencies. We further establish that, of the two isoforms of the Kv3.1 potassium channel that are expressed in these neurons, Kv3.1a and Kv3.1b, the decrease in Kv3.1 current is mediated by selective phosphorylation of the Kv3.1b isoform. Using site-directed mutagenesis, we identify a specific C-terminal phosphorylation site responsible for the observed difference in response of the two isoforms to protein kinase C activation. Our results suggest that modulation of Kv3.1 by phosphorylation allows auditory neurons to tune their responses to different patterns of sensory stimulation.

Two Mechanisms for Human Immunodeficiency Virus Type 1 Inhibition by N-Terminal Modifications of RANTES

C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a C-terminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.

造血幹細胞数および造血能を制御する抑制性アダプター蛋白質Ｌｎｋ

Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells, and are responsible for blood production throughout adult life. Recent studies have demonstrated the potential of HSCs to produce various nonhematopoietic cells. Amplification of HSCs represents a potentially powerful approach not only to the treatment of various blood disorders but also to applying gene therapy or to regenerative medicine. Lnk is an adaptor protein consisting of PH, SH2 domains, and a C-terminal tyrosine phosphorylation site. Marked expansion of Blineage cells occurs in lnk-/- mice, indicating Lnk regulates B cell production by negatively controlling growth signals through c-Kit in B cell precursors. Lnk is also expressed in hematopoietic progenitors, and the number and the hematopoietic ability of HSCs are significantly increased in the absence of Lnk. While augmented signaling through c-Kit partly contributed to the increased progenitor cells in lnk-/- mice, it is likely that there exist Lnkdependent but c-Kit-independent signaling pathways whose absence leads to augmented ability of lnk-/- HSCs. These indicate that Lnk plays critical roles in the expansion and function of HSCs and provide useful clues for the amplification of HSCs and progenitor cells.

Intracellular trafficking of human CXCR1 and CXCR2: regulation by receptor domains and actin-related kinases.

In this study we investigated the regulation of CXCR1 and CXCR2 intracellular trafficking. First, we produced a chimeric CXCR2 receptor that contained the internalization motifs of both CXCR2 and CXCR1 (CXCR2: LLKIL sequence; CXCR1: C-terminal phosphorylation sites). Elevated levels of internalization were induced by different ELR-expressing CXC chemokines on the chimeric receptor, as compared to wild-type CXCR2. Analysis of inter-relationships between CXCR1 and CXCR2 during internalization indicated that the exposure of cells that expressed both CXCR1 and CXCR2 to CXCL8 or CXCL6 resulted in decreased levels of CXCR1 internalization as compared to those in cells that expressed only CXCR1. To characterize the role of actin-related components in CXCR1 and CXCR2 trafficking, wortmannin, a potent inhibitor of phosphatidylinositol kinases, was used. The presence of wortmannin during receptor recycling inhibited CXCR1 and CXCR2 re-expression following CXCL8-induced internalization, and resulted in a marked disruption of the proper organization of actin filaments. The kinase-dependent recycling process required CXCR2 C-terminal phosphorylation sites. Our results suggest that actin-related kinases are required for the proper functionality of actin filaments, which are the instrumental factors needed for receptor recycling. In all, CXCR1 and CXCR2 internalization and recycling are tightly regulated by receptor domains and by actin-related kinases.

Membrane Localization of Src Homology 2-Containing Inositol 5′-Phosphatase 2 via Shc Association Is Required for the Negative Regulation of Insulin Signaling in Rat1 Fibroblasts Overexpressing Insulin Receptors

Lipid phosphatase SHIP2 [Src homology 2 (SH2)-containing inositol 5'-phosphatase 2] has been shown to be a physiologically critical negative regulator of insulin signaling. We investigated the molecular mechanism by which SHIP2 negatively regulates insulin-induced phosphorylation of Akt, a key downstream molecule of phosphatidylinositol 3-kinase important for the biological action of insulin. Overexpression of wild-type SHIP2 (WT-SHIP2) inhibited insulin-induced phosphorylation of Akt at both Thr(308) and Ser(473) in Rat1 fibroblasts expressing insulin receptors. The degree of inhibition was less in the cells expressing either a mutant SHIP2 with R47Q change (R/Q-SHIP2) in the SH2 domain, or a mutant SHIP2 with Y987F change (Y/F-SHIP2) in the C-terminal tyrosine phosphorylation site. However, on addition of a myristoylation signal, WT-SHIP2, R/Q-SHIP2, and Y/F-SHIP2 all efficiently inhibited insulin-induced Akt phosphorylation at both residues, whereas a 5'-phosphatase-defective mutant SHIP2 (deltaIP-SHIP2) with the myristoylation signal did not. Interestingly, the degree of inhibition of Akt phosphorylation by R/Q-SHIP2 and Y/F-SHIP2 is well correlated with the extent of their association with Shc. In addition, overexpression of WT-Shc increased the insulin-induced association of SHIP2 with Shc, whereas a decrease in the amount of Shc on expression of antisense Shc mRNA led to a reduction in the SHIP2-Shc association. Furthermore, the inhibitory effect on insulin-induced Akt phosphorylation by WT-SHIP2 was decreased in antisense-Shc cells. These results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts.

E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis.

It has been proposed that the E2F1 transcription factor serves as a link between the Rb/E2F proliferation pathway and the p53 apoptosis pathway by inducing the expression of p19ARF, a protein that regulates p53 stability. We find that although p19ARF contributes to p53 accumulation in response to E2F expression, p19ARF is not required for E2F1-mediated apoptosis. E2F1 can signal p53 phosphorylation in the absence of p19ARF, similar to the observed modifications to p53 in response to DNA damage. These modifications are not observed in the absence of p19ARF following expression of E2F2, an E2F family member that does not induce apoptosis in mouse embryo fibroblasts but can induce p19ARF and p53 protein expression. p53 modification is found to be crucial for E2F1-mediated apoptosis, and this apoptosis is compromised when E2F1 is coexpressed with a p53 mutant lacking many N- and C-terminal phosphorylation sites. Additionally, E2F1-mediated apoptosis is abolished in the presence of caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases that phosphorylate p53. These findings suggest that p53 phosphorylation is a key step in E2F1-mediated apoptosis and that this modification can occur in the absence of p19ARF.

Identification of a Region within the ErbB2/HER2 Intracellular Domain That Is Necessary for Ligand-independent Association

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.

Characterization of PDK2 activity against protein kinase B gamma.

Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBgamma identified protein kinase C (PKC) zeta, an atypical PKC, as an interactor with PKBgamma, an association requiring the pleckstrin homology domain of PKBgamma. Endogenous PKBgamma was shown to associate with endogenous PKCzeta both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCzeta, whether endogenous PKCzeta from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCzeta from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBgamma, in vitro. In vivo, overexpression of PKCzeta stimulated the phosphorylation of approximately 50% of the PKBgamma molecules, suggesting a physiologically meaningful effect. However, pure PKCzeta protein was incapable of phosphorylating S472 of PKBgamma. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCzeta immunoprecipitates. Staurosporine inhibited the PKCzeta activity but not the PDK2 activity in PKCzeta immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCzeta and that PKCzeta, by binding PKBgamma, functions to deliver the PDK2 to a required location. PKCzeta thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBgamma. Because both PKCzeta and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCzeta, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.

Protein Kinase C Phosphorylation of Purified Na,K-ATPase: C-Terminal Phosphorylation Sites at the α- and γ-Subunits Close to the Inner Face of the Plasma Membrane

The α-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the α-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents. Two novel phosphorylation sites are possible candidates for this PKC phosphorylation: Thr-938 in the M8/M9 loop located very close to the PKA site, and Ser-774, in the proximal part of the M5/M6 hairpin. Both sites are highly conserved in all known α-subunits, indicating a physiological role. A similar pattern of detergent-mediated phosphorylation by PKC was found in pig kidney Na,K-ATPase α-subunit. Interestingly, the kidney-specific γ-subunit was phosphorylated by PKC in the presence of detergent. The close proximity of the novel PKC sites to the membrane suggests that targeting proteins to tether PKC into the membrane phase is important in controlling the in vivo phosphorylation of this novel class of membrane-adjacent PKC sites. It is suggested that in purified preparations where functional targeting may be impaired detergents are needed to expose the sites.

Novel alternative splice variants of rat phosphodiesterase 7B showing unique tissue-specific expression and phosphorylation.

cDNA species coding for novel variants of cyclic-AMP-specific phosphodiesterases (PDEs), namely the PDE7B family, were isolated from rats and characterized. Rat PDE7B1 (RNPDE7B1) was composed of 446 amino acid residues. Rat PDE7B2 (RNPDE7B2) and PDE7B3 (RNPDE7B3), which possessed unique N-terminal sequences, consisted of 359 and 459 residues respectively. Northern hybridization analysis showed that rat PDE7B transcripts were particularly abundant in the striatum and testis. PCR analyses revealed that rat PDE7B2 transcripts were restricted to the testis and that low levels of PDE7B3 transcripts were expressed in the heart, lung and skeletal muscle. In situ hybridization analysis demonstrated that rat PDE7B transcripts were expressed in striatal neurons and spermatocytes. In spermatocytes, rat PDE7B transcripts were expressed in a stage-specific manner during spermatogenesis. The K(m) values of recombinant rat PDE7B1, PDE7B2 and PDE7B3 for cAMP were 0.05, 0.07 and 0.05 microM respectively. Each rat PDE7B variant was the most sensitive to 3-isobutyl-1-methylxanthine (IC(50) 1.5-2.1 microM). Two phosphorylation sites for cAMP-dependent protein kinase (PKA) were found in rat PDE7B1 and PDE7B3, whereas rat PDE7B2 possessed one site. PKA-dependent phosphorylation was observed in C-terminal phosphorylation sites of three rat PDE7B variants, in addition to unique N-terminal regions of rat PDE7B1 and PDE7B3. Unique tissue distribution and PKA-dependent phosphorylation of PDE7B variants suggested that each variant has a specific role for cellular functions via cAMP signalling in various tissues.

Novel alternative splice variants of rat phosphodiesterase 7B showing unique tissue-specific expression and phosphorylation

cDNA species coding for novel variants of cyclic-AMP-specific phosphodiesterases (PDEs), namely the PDE7B family, were isolated from rats and characterized. Rat PDE7B1 (RNPDE7B1) was composed of 446 amino acid residues. Rat PDE7B2 (RNPDE7B2) and PDE7B3 (RNPDE7B3), which possessed unique N-terminal sequences, consisted of 359 and 459 residues respectively. Northern hybridization analysis showed that rat PDE7B transcripts were particularly abundant in the striatum and testis. PCR analyses revealed that rat PDE7B2 transcripts were restricted to the testis and that low levels of PDE7B3 transcripts were expressed in the heart, lung and skeletal muscle. In situ hybridization analysis demonstrated that rat PDE7B transcripts were expressed in striatal neurons and spermatocytes. In spermatocytes, rat PDE7B transcripts were expressed in a stage-specific manner during spermatogenesis. The Km values of recombinant rat PDE7B1, PDE7B2 and PDE7B3 for cAMP were 0.05, 0.07 and 0.05μM respectively. Each rat PDE7B variant was the most sensitive to 3-isobutyl-1-methylxanthine (IC50 1.5–2.1μM). Two phosphorylation sites for cAMP-dependent protein kinase (PKA) were found in rat PDE7B1 and PDE7B3, whereas rat PDE7B2 possessed one site. PKA-dependent phosphorylation was observed in C-terminal phosphorylation sites of three rat PDE7B variants, in addition to unique N-terminal regions of rat PDE7B1 and PDE7B3. Unique tissue distribution and PKA-dependent phosphorylation of PDE7B variants suggested that each variant has a specific role for cellular functions via cAMP signalling in various tissues.

The defective transforming phenotype of c-Jun Ala(63/73) is rescued by mutation of the C-terminal phosphorylation site.

Cotransfection of primary rat embryo fibroblasts (REF) with c-Jun and activated Ras leads to oncogenic transformation and this process requires the phosphorylation of the N-terminal domain of c-Jun. Ras augments this phosphorylation and, consequently activates the c-Jun transactivation property of TRE (TPA Responsive Element)-dependent promoters. To analyse the role of the c-Jun C-terminal phosphorylation site in oncogenic cooperation we tested the activities of N-terminal c-Jun Ala(63/73) (named Nt), C-terminal c-Jun Ala(234/242/246/252) (named Ct) and (Nt+Ct)-with both mutations-non-phosphorylatable c-Jun mutants. In cooperation with Ras, the Ct mutant and wt c-Jun display similar oncogenic properties whereas the Nt form was defective in transforming REF cells. Unexpectedly, the Nt+Ct mutant exhibited identical oncogenic properties to wt c-Jun, demonstrating that the Ct mutation rescues in cis the Nt mutation. The transcriptional activity and the capacity to bind the c-Jun coactivator CREB Binding Protein (CBP) were enhanced by Ras for the wt and Ct proteins but not for the Nt mutant. Interestingly, the Nt+Ct mutant presents identical transactivation and CBP binding activities to wt c-Jun. Therefore the rescue in cis of the defective Nt mutation by the Ct mutation seems to be due to the recovery of CBP binding. Our results revealed that the process of oncogenic cooperation can occur between Ras and the Nt+Ct non-phosphorylatable c-Jun protein.

Functional effects of APS and SH2-B on insulin receptor signalling.

APS [for 'adapter protein with a pleckstrin homology (PH) and Src homology 2 (SH2) domain'] belongs to a family of adapter proteins involved in signalling by the receptors for insulin, insulin-like growth factor 1, platelet-derived growth factor and nerve growth factor. Other members include alternatively spliced SH2-B isoforms (SH2Balpha, SH2-Bbeta and SH2-Bgamma) and Lnk. These have a C-terminal SH2 domain, a central PH domain and an N-terminal proline-rich region. SH2Balpha, APS and Lnk have a conserved C-terminal tyrosine phosphorylation site, whereas the alternatively spliced SH2-Bbeta and SH2-Bgamma have distinct C-termini. There is considerable sequence similarity between APS, SH2-B and Lnk, particularly in the SH2 domain. Both APS and SH2-Balpha interact with the insulin-receptor activation loop phosphorylation sites and undergo insulin-stimulated tyrosine phosphorylation, although the phosphorylation of SH2-B is considerably weaker. APS couples c-Cbl to the insulin receptor, resulting in ubiquitination of the insulin receptor. We established cell lines [Chinese hamster ovary (CHO). T-APS and CHO. T-SH2-B cells] overexpressing APS and SH2-Balpha to study their roles in insulin receptor signalling. Either adapter protein enhances insulin receptor and ERK (extracellular-signal-regulated kinase) phosphorylation. In CHO. T-APS cells, Akt phosphorylation is observed earlier than in CHO.T-SH2-B cells. Both enhance insulin-stimulated Akt activation but APS seems to cause greater activation. Thus APS and SH2-B have similar effects on insulin receptor signalling, although the effects of SH2-B are independent of its phosphorylation.

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Ribosomal S6 Kinase 2 Inhibition by a Potent C-terminal Repressor Domain Is Relieved by Mitogen-activated Protein-Extracellular Signal-regulated Kinase Kinase-regulated Phosphorylation

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.

The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization.

Smad proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.