The serine/threonine kinase Pim-1

The human pim-1 gene encodes a serine/threonine kinase, which belongs to the group of calcium/calmodulin-regulated kinases (CAMK). It contains a characteristic kinase domain, a so-called ATP anchor and an active site. In mouse and human, two Pim-1 proteins are produced from the same gene by using an alternative upstream CUG initiation codon, a 44 kD and another, shorter 34 kD form that both contain the kinase domain. Expression of Pim-1 is widespread and ranges from the hematopoietic and lymphoid system to prostate, testis and oral epithelial cells. Two other proteins with significant sequence similarities exist, Pim-2 and Pim-3; both are also serine/threonine kinases and have largely overlapping functions. Pim-1 is able to phosphorylate different targets, most of which are involved in cell cycle progression or apoptosis. Pim-1 expression can be induced by several external stimuli in particular by a number of cytokines relevant in the immune system, which led to the labeling of Pim-1 as a "booster" for the immune response.     

Identification and cloning of a CD43-associated serine/threonine kinase

CD43 is expressed on most hematopoetic cells and has been shown to regulate the activation and adhesion of T cells. We have cloned a serine/threonine kinase that can interact with the cytoplasmic domain of CD43. This protein is expressed in multiple tissues, including lymphoid cells. Analysis of the subcellular localization reveals it to be present in both the nucleus and the cytoplasm of the cell. The identification of this protein suggests that CD43 may mediate its biologic effects through activation of a kinase cascade, resulting in the regulation of cell growth.     

Neuronal functions of the novel serine/threonine kinase Ndr2

We have identified a novel member of the Ndr subfamily of serine/threonine protein kinases, Ndr2, as a gene product that is induced in the mouse amygdala during fear memory consolidation and examined a possible function of this kinase in neural differentiation. Expression of Ndr2 mRNA was detected in various cortical and subcortical brain regions, as well as non-neuronal tissues. Its expression in the amygdala was increased 6 h after Pavlovian fear conditioning training and returned to control levels within 24 h. To study intracellular localization and functions of Ndr2, EGFP::Ndr2 fusion proteins were expressed in rat pheochromocytoma (PC12) cells and acutely isolated cortical neurons, thereby revealing an association of Ndr2 with the actin cytoskeleton in somata, neurites and filopodia, in spines and at sites of cell contact. Co-precipitation and pull-down experiments support this finding. Evidence for an involvement of Ndr2 in actin-mediated cellular functions further comes from the observation of decreased cell spreading and changes in neurite outgrowth that were associated with protein serine phosphorylation in transfected PC12 cells. Together, our data suggest that Ndr2 is an interesting candidate gene for the regulation of structural processes in differentiating and mature neuronal cells.     

The serine threonine kinase RIP3: lost and found

Receptor-interacting protein kinase-3 (RIP3, or RIPK3) is an essential protein in the “programmed”, or “regulated” necrosis cell death pathway that is activated in response to death receptor ligands and other types of cellular stress. Programmed necrotic cell death is distinguished from its apoptotic counterpart in that it is not characterized by the activation of caspases; unlike apoptosis, programmed necrosis results in plasma membrane rupture, thus spilling the contents of the cell and triggering the activation of the immune system and inflammation. Here we discuss findings, including our own recent data, which show that RIP3 protein expression is absent in many cancer cell lines. The recent data suggests that the lack of RIP3 expression in a majority of these deficient cell lines is due to methylation-dependent silencing, which limits the responses of these cells to pro-necrotic stimuli. Importantly, RIP3 expression may be restored in many cancer cells through the use of hypomethylating agents, such as decitabine. The potential implications of loss of RIP3 expression in cancer are explored, along with possible consequences for chemotherapeutic response. [BMB Reports 2015; 48(6): 303-312]     

The serine/threonine kinase HIPK2 interacts with TRADD, but not with CD95 or TNF-R1 in 293T cells

HIPK2 has been described as a homoedomain-interacting protein kinase with a nuclear localization. Here we describe that HIPK2 can also associate with TRADD, a protein that interacts with tumor necrosis factor receptor type 1 (TNF-R1). Under the conditions where HIPK2/TRADD association was found, no direct interaction of HIPK2 with CD95, TNF-R1, FADD or caspase-8 could be detected. Therefore, HIPK2 may play a role in TNF-R1 mediated signaling.     

The alphaherpesvirus serine/threonine kinase us3 disrupts promyelocytic leukemia protein nuclear bodies

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection, including preventing virus-induced apoptosis, facilitating nuclear egress of capsids, stimulating mRNA translation and promoting cell-to-cell spread of virus infection. Given this diversity, the full spectrum of Us3 function may not yet be recognized. We noted, in transiently transfected cells, that herpes simplex virus type 2 (HSV-2) Us3 disrupted promyelocytic leukemia protein nuclear bodies (PML-NBs). However, PML-NB disruption was not observed in cells expressing catalytically inactive HSV-2 Us3. Analysis of PML-NBs in Vero cells transfected with pseudorabies virus (PRV) Us3 and those in Vero cells infected with Us3-null or -repaired PRV strains indicated that PRV Us3 expression also leads to the disruption of PML-NBs. While loss of PML-NBs in response to Us3 expression was prevented by the proteasome inhibitor MG132, Us3-mediated degradation of PML was not observed in infected cells or in transfected cells expressing enhanced green fluorescent protein (EGFP)-tagged PML isoform IV. These findings demonstrate that Us3 orthologues derived from distantly related alphaherpesviruses cause a disruption of PML-NBs in a kinase- and proteasome-dependent manner but, unlike the alphaherpesvirus ICP0 orthologues, do not target PML for degradation.     

Crystal structure of aurora-2, an oncogenic serine/threonine kinase

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases.     

The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB

The serine/threonine kinase Akt, or protein kinase B (PKB), has recently been a focus of intense research. It appears that Akt/PKB lies in the crossroads of multiple cellular signaling pathways and acts as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI 3-kinase). Akt/PKB is particularly important in mediating several metabolic actions of insulin. Another major activity of Akt/PKB is to mediate cell survival. In addition, the recent discovery of the tumor suppressor PTEN as an antagonist of PI 3-kinase and Akt/PKB kinase activity suggests that Akt/PKB is a critical factor in the genesis of cancer. Thus, elucidation of the mechanisms of Akt/PKB regulation and its physiological functions should be important for the understanding of cellular metabolism, apoptosis, and cancer.     

Cmk2, a novel serine/threonine kinase in fission yeast

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Cloning and characterization of AWP1, a novel protein that associates with serine/threonine kinase PRK1 in vivo

We describe the cloning and expression of cDNAs encoding a novel human protein of 208 amino acid residues with a predicted molecular mass of 22.6kDa and its mouse homologue. We name this protein as AWP1 (associated with PRK1). AWP1 is a ubiquitously expressed protein, and the Awp1 gene is switched on during early human and mouse development. When expressed in COS-1 cells, the Myc-tagged AWP1 has an apparent molecular mass higher than that deduced from its amino acid sequence. AWP1 possesses a conserved zf-A20 zinc finger domain at its N-terminal and a zf-AN1 zinc finger domain at its C-terminal. Co-immunoprecipitation experiments revealed that mouse AWP1 specifically interacts with a rat serine/threonine protein kinase PRK1 in vivo. Hence, AWP1 may play a regulatory role in mammalian signal transduction pathways.     

Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.     

Negative regulation of the serine/threonine kinase B-Raf by Akt

B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.     

Effective T-cell immune responses in the absence of the serine/threonine kinase RIP2

The serine/threonine kinase RIP2 has been reported to be essential for Nod1 and Nod2 mediated cell activation, and has been suggested to play a role in the signaling cascade downstream of the T-cell receptor. We sought to ascertain the exact role of RIP2 in T-helper cell differentiation and CD8+ T-cell effector function in vivo and in vitro. In contrast to previous reports, we found that RIP2-deficient T cells did not exhibit impaired proliferation upon TCR engagement in vitro, and differentiation to cytokine producing Th1 or Th2 cells was normal in the absence of RIP2. These results were confirmed in vivo, as wild-type and RIP2-deficient virus-specific CD8+ T cells expanded comparably in mice after LCMV infection. Wild-type and RIP2-deficient CD4+ and CD8+ T cells from infected mice also showed similar proliferation and cytokine production when restimulated with full or partial agonist peptides ex vivo. Furthermore, no significant difference in adaptive T-cell responses could be observed between wild-type and RIP2-deficient mice after Listeria monocytogenes infection. Thus contrary to early reports, our data show that RIP2 is not an essential component of the TCR signaling machinery.     

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 ? resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.     

Autoregulatory characteristics of a Bacillus anthracis serine/threonine kinase

BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.     

Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.