Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> growth

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.Received 15 January 2004; received after revision 20 February 2004; accepted 4 March 2004     

A “SYDE” effect of hierarchical phosphorylation: possible relevance to the cystic fibrosis basic defect

The motif “SYDE”, incorporating the protein kinase CK2 consensus sequence (S-x-x-E) has been found to be phosphorylated at both its serine and tyrosine residues in several proteins. Of special interest is the case of cystic fibrosis Transmembrane-conductance Regulator (CFTR), where this motif is close to the residue (F508), whose deletion is the by far commonest cause of cystic fibrosis. Intriguingly, however, CFTR S511 cannot be phosphorylated by CK2 to any appreciable extent. Using a number of peptide substrates encompassing the CFTR “SYDE” site we have recently shown that: (1) failure of CK2 to phosphorylate the S⁵¹¹YDE motif is due to the presence of Y512; (2) CK2 readily phosphorylates S511 if Y512 is replaced by a phospho-tyrosine; (3) the Src family protein tyrosine kinase Lyn phosphorylates Y512 in a manner that is enhanced by the deletion of F508. These data, in conjunction with the recent observation that by inhibiting CK2 the degradation of F508delCFTR is reduced, lead us to hypothesize that the hierarchical phosphorylation of the motif SYDE by the concerted action of protein tyrosine kinases and CK2 is one of the mechanisms that cooperate to the premature degradation of F508delCFTR.     

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.     

Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner

During mitosis, cells detach, and the cell–matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.     

Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells

Akt (PKB) is a critical kinase in cell-survival pathways. Its activity depends on the phosphorylation of Thr308 and Ser473, by PDK1 and mTORC2, respectively. We found that Akt can be further stimulated through phosphorylation of Ser129 by another kinase, CK2. Here we show that phosphorylation of Akt at Ser129 also facilitates its association with Hsp90 chaperone, thus preventing Thr308 dephosphorylation. This is supported by the following observations: (1) phospho-Thr308 decreases when Ser129 is mutated to alanine, (2) this decrease is abolished by cell treatment with okadaic acid (to inactivate PP2A) or geldanamycin (to inactivate Hsp90), (3) phosphorylation of Ser129 neither enhances the activity of PDK1 nor hampers the in vitro activity of PP2A on Thr308, but increases the Hsp90 association to Akt. These data support the view that the antiapoptotic potential of CK2 is at least in part mediated by its ability to maintain Akt in its active form.     

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO^? present in TBCB, which is similar to the EEY/F-COO^? element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.     

Targeting of the Akt/PKB kinase to the actin skeleton

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.Received 10 September 2003; accepted 25 September 2003     

Mitotic regulation of the anaphase-promoting complex

Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C), a large multiprotein E3 ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome. APC/C has two activating subunits, Cdc20 and Cdh1. The well-established view is that Cdc20 activates APC/C from the onset of mitosis through the metaphase-anaphase transition, and that Cdh1 does so from anaphase through G1. Recent work, however, indicates that Cdh1 also activates APC/C in early mitosis and that this APC/C pool targets the anaphase inhibitor securin. To prevent premature degradation of securin, the nuclear transport factors Nup98 and Rae1 associate with APC/C^Cdh1-securin complexes. In late metaphase, when all kinetochores are attached to spindle microtubules and the spindle assembly checkpoint is satisfied, Nup98 and Rae1 are released from these complexes, thereby allowing for prompt ubiquitination of securin by APC/C^Cdh1. This, and other mechanisms by which the catalytic activity of APC/C is tightly regulated to ensure proper timing of degradation of each of its mitotic substrates, are highlighted. Received 8 October 2006; received after revision 24 November 2006; accepted 8 January 2007     

Polo-box domain: a versatile mediator of polo-like kinase function

Members of the polo subfamily of protein kinases have emerged as important regulators in diverse aspects of the cell cycle and cell proliferation. A large body of evidence suggests that a highly conserved polo-box domain (PBD) present in the C-terminal non-catalytic region of polo kinases plays a pivotal role in the function of these enzymes. Recent advances in our comprehension of the mechanisms underlying mammalian polo-like kinase 1 (Plk1)-dependent protein–protein interactions revealed that the PBD serves as an essential molecular mediator that brings the kinase domain of Plk1 into proximity with its substrates, mainly through phospho-dependent interactions with its target proteins. In this review, current understanding of the structure and functions of PBD, mode of PBD-dependent interactions and substrate phosphorylation, and other phospho-independent functions of PBD are discussed.     

Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82

A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> possesses a unique dual-specificity serine/threonine and tyrosine kinase,Pfnek3

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg²⁺ and Mn²⁺ cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3’s dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.     

Phosphorylation-dependent prolyl isomerization: a novel signaling regulatory mechanism

Protein phosphorylation on serine or threonine residues preceding proline (Ser/Thr-Pro) plays an essential role for regulating various cellular processes, including cell cycle progression. Although phosphorylation has been proposed to regulate the function of a protein by inducing conformational changes, much less is known about what phosphate additions actually do and how the functions of phosphoproteins are coordinated. Proline is important for determining protein structure because it exists in cis or trans conformation and can put kinks into a polypeptide chain. We have shown that phosphorylation on Ser/Thr-Pro motifs reduces the cis/trans isomerization rate of Ser/Thr-Pro bonds. At the same time, proteins containing phosphorylated Ser/Thr-Pro motifs are substrates for the prolyl isomerase Pin1. The WW domain of Pin1 acts as a phosphoserine/threonine-binding module binding a defined subset of mitosis-specific phosphoproteins, such as Cdc25 and tau. These interactions target the enzymatic activity of Pin1 close to its substrates. In contrast to other prolyl isomerases (peptidyl-prolyl isomerases, PPlases), Pin1 has an extremely high degree of substrate specificity, specifically isomerizing phosphorylated Ser/Thr-Pro bonds. Therefore, Pin1 binds and regulates the function of a defined subset of phosphoproteins. Furthermore, inhibiting Pin1 function is lethal for dividing cells. Interestingly, Pin1, which can restore the biological function of phosphorylated tau, is sequestered in the neurofibrillary tangles in Alzheimer's brains. Thus, we have proposed a novel signaling regulatory mechanism, where protein phosphorylation creates binding sites for Pin1, which can then latch on to and isomerize the phosphorylated Ser/Thr-Pro peptide bond. In turn, this may change the shape of the protein, regulating its activity, dephosphorylation, degradation or location in the cell. This new post-phosphorylation regulatory mechanism appears to play an important role in normal cell function, such as mitotic progression, and in the pathogenesis of some human pathologies, such as Alzheimer's disease.     

Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB

p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.     

Role of Sam68 as an adaptor protein in signal transduction

Sam68, the substrate of Src in mitosis, belongs to the family of RNA binding proteins. Sam68 contains consensus sequences to interact with other proteins via specific domains. Thus, Sam68 has various proline-rich sequences to interact with SH3 domain-containing proteins. Moreover, Sam68 also has a C-terminal domain rich in tyrosine residues that is a substrate for tyrosine kinases. Tyrosine phosphorylation of Sam68 promotes its interaction with SH2 containing proteins. The association of Sam68 with SH3 domain-containing proteins, and its tyrosine phosphorylation may negatively regulate its RNA binding activity. The presence of these consensus sequences to interact with different domains allows this protein to participate in signal transduction pathways triggered by tyrosine kinases. Thus, Sam68 participates in the signaling of T cell receptors, leptin and insulin receptors. In these systems Sam68 is tyrosine phosphorylated and recruited to specific signaling complexes. The participation of Sam68 in signaling suggests that it may function as an adaptor molecule, working as a dock to recruit other signaling molecules. Finally, the connection between this role of Sam68 in protein-protein interaction with RNA binding activity may connect signal transduction of tyrosine kinases with the regulation of RNA metabolism.Received 16 July 2004; received after revision 12 August 2004; accepted 18 August 2004     

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130^Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130^Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect. Received 21 January 2008; received after revision 4 March 2008; accepted 19 March 2008     

Recruitment of Pyk2 to SHPS-1 signaling complex is required for IGF-I-dependent mitogenic signaling in vascular smooth muscle cells

In vascular smooth muscle cells, IGF-I stimulates SHPS-1/SHP2/Src complex formation which is required for IGF-I-stimulated cell proliferation. Using SHP2/Src silencing and a Pyk2/Y402F mutant, we showed that Pyk2 was also recruited to the SHPS-1 complex. Pyk2 recruitment to SHPS-1 is mediated via the interaction of Pyk2 Tyr402 and the Src in response to IGF-I. Following Src/Pyk2 association, Src phosphorylates Pyk2 on Tyr881 providing a binding site for Grb2. Cells expressing Pyk2/Y881F showed decreased Grb2 recruitment to SHPS-1 and impaired Shc/Grb2 association. This change led to reduced Erk1/2 (MAP kinase) activation and cell proliferation in response to IGF-I. Our results show that, following its recruitment to the SHPS-1 signaling complex, Pyk2 localizes Grb2 in close proximity to Shc thereby facilitating Shc/Grb2 association which leads to Erk1/2 activation in response to IGF-I. Thus, Pyk2 recruitment to SHPS-1 plays an important role in regulating the IGF-I-stimulated mitogenic response.