Murine Protein Kinase Research Articles

The Antagonism between the Murine Gammaherpesvirus Protein Kinase and Global Interferon Regulatory Factor 1 Expression Shapes the Establishment of Chronic Infection.

Gammaherpesviruses infect most vertebrate species and are associated with B cell lymphomas. Manipulation of B cell differentiation is critical for natural infection and lymphomagenesis driven by gammaherpesviruses. Specifically, human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) drive differentiation of infected naive B cells into the germinal center to achieve exponential increase in the latent viral reservoir during the establishment of chronic infection. Infected germinal center B cells are also the target of viral lymphomagenesis, as most EBV-positive B cell lymphomas bear the signature of the germinal center response. All gammaherpesviruses encode a protein kinase, which, in the case of Kaposi's sarcoma-associated herpesvirus (KSHV) and MHV68, is sufficient and necessary, respectively, to drive B cell differentiation in vivo. In this study, we used the highly tractable MHV68 model of chronic gammaherpesvirus infection to unveil an antagonistic relationship between MHV68 protein kinase and interferon regulatory factor 1 (IRF-1). IRF-1 deficiency had minimal effect on the attenuated lytic replication of the kinase-null MHV68 in vivo. In contrast, the attenuated latent reservoir of the kinase-null MHV68 was partially to fully rescued in IRF-1-/- mice, along with complete rescue of the MHV68-driven germinal center response. Thus, the novel viral protein kinase-IRF-1 antagonism was largely limited to chronic infection dominated by viral latency and was less relevant for lytic replication during acute infection and in vitro. Given the conserved nature of the viral and host protein, the antagonism between the two, as defined in this study, may regulate gammaherpesvirus infection across species. IMPORTANCE Gammaherpesviruses are prevalent pathogens that manipulate physiological B cell differentiation to establish lifelong infection. This manipulation is also involved in gammaherpesvirus-driven B cell lymphomas, as differentiation of latently infected B cells through the germinal center response targets these for transformation. In this study, we define a novel antagonistic interaction between a conserved gammaherpesvirus protein kinase and a host antiviral and tumor suppressor transcription factor. The virus-host antagonism unveiled in this study was critically important to shape the magnitude of gammaherpesvirus-driven germinal center response. In contrast, the virus-host antagonism was far less relevant for lytic viral replication in vitro and during acute infection in vivo, highlighting the emerging concept that nonoverlapping mechanisms shape the parameters of acute and chronic gammaherpesvirus infection.

Vitamin D Inhibits Monocyte/macrophage Pro-inflammatory Cytokine Production by Targeting Mitogen-Activated Protein Kinase Phosphatase-1

Vitamin D deficiency has been linked to severity of various allergic diseases. However, the mechanisms of vitamin D-mediated inhibition of inflammation remain poorly understood. In this study, we investigated the inhibitory effects of physiologic levels of vitamin D on lipopolysaccharide (LPS)-stimulated immune responses. Peripheral blood mononuclear cells (PBMC) from normal and asthmatic subjects, and murine bone marrow-derived macrophages (BMM) were pre-incubated with 1,25(OH)2D3 or 25(OH)D3, followed by LPS stimulation. p38 phosphorylation was detected by flow cytometry and Western Blotting. mRNA expression was analyzed by real-time PCR. Vitamin D receptor (VDR) binding and histone acetylation at vitamin D response element in the murine mitogen-activated protein kinase phosphatase-1 (MKP-1) promoter were detected by Chromatin Immunoprecipitation. Physiologic concentrations of 1,25(OH)2D3, and 25(OH)D3 inhibited LPS induced p38 phosphorylation and IL-6 production in normal subjects by 78% (p<0.01, n=4) and 77% (p<0.01, n=4) respectively. 1,25(OH)2D3 inhibited LPS induced p38 phosphorylation in asthmatic subjects by 57% (p<0.01, n=5). No inhibition of LPS induced p38 phosphorylation was found at 15ng/ml of 25(OH)D3. 1,25(OH)2D3 significantly up-regulated the expression of MKP-1 mRNA in adherent PBMC which consisted mainly of monocytes (2.2±0.2 fold, p<0.01, n=5) and BMM (2.6 ± 0.2 fold, p<0.01, n=3), increased binding of VDR (3.7±0.4 fold, p<0.05, n=3) and histone H4 acetylation (6.26±0.04 fold, p<0.05, n=3) at MKP-1 promoter. In BMM from MKP1-/- mice, the inhibition of LPS-induced p38 phosphorylation by vitamin D was completely abolished. This study demonstrates that up-regulation of MKP-1 by vitamin D inhibits LPS-induced p38 activation and cytokine production in monocytes/macrophages.

Identification and expression analysis of three novel splice variants of protein kinase A catalytic β subunit gene in the mouse using combinatorial in silico and molecular biology approaches

The murine, bovine and human protein kinase A catalytic β (Cβ) subunit genes encode several splice variants. Ten human Cβ gene splice variants, namely Cβ1, Cβ2, Cβ3, Cβ3b, Cβ3ab, Cβ3abc, Cβ4, Cβ4b, Cβ4ab and Cβ4abc, have been reported. Four splice variants of the murine Cβ gene are homologues of human Cβ1, Cβ2, Cβ3 and Cβ4 variants. Using a combinatorial approach comprising bioinformatics tools and molecular biology techniques, we have identified three novel alternatively spliced transcript variants of the mouse Cβ gene designated Cβ5, Cβ6 and Cβ7. These transcript variants differ in their first coding exon. New exons of Cβ5 and Cβ6 variants can encode different N-terminals; however, the new exon of variant Cβ7, when spliced with exon 2, encountered a stop codon in all three reading frames and thus cannot form a functional protein. The Cβ6 variant showed an ubiquitous pattern of expression, whereas Cβ5 was not expressed in muscle tissue on postnatal days (PN)3 and 15. Also, Cβ7 was found to be expressed only in brain and muscle tissues at PN3 and was absent in all tissues examined at PN15 and PN60. The post-translational modification analysis showed characteristic signature sequence motifs in predicted proteins important for the functionality of the protein. The human homologues of variants reported in the present study have not yet been identified. The present study reports three novel splice variants that have not been identified using conventional approaches of alternative splice variant detection methods. Therefore, the approach used in the present study can be used to identify splice variants of genes in organisms. Database GenBank accession numbers: JN189786, JN189787 and JN189788.

A Conserved Glu–Arg Salt Bridge Connects Coevolved Motifs That Define the Eukaryotic Protein Kinase Fold

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG–αH–αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K m(ATP) and a decrease in k cat. Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208–Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.

Disruption of the Murine Protein Kinase Cβ Gene Promotes Gallstone Formation and Alters Biliary Lipid and Hepatic Cholesterol Metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.

TGF-β1/ALK5-induced monocyte migration involves PI3K and p38 pathways and is not negatively affected by diabetes mellitus

Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. Transforming growth factor β1 (TGF-β1) mediates monocyte motility and stimulates arteriogenesis. TGF-β1 signalling mechanisms mediating monocyte motility are unknown so far. Moreover, the influence of cardiovascular risk factor diabetes on TGF-β1-induced monocyte migration remains to be elucidated. Stimulation of primary human monocytes with TGF-β1 endorsed phosphorylation of v-Akt murine thymoma viral oncogene analogues protein (AKT), p38, and extracellular signal-related kinase 1/2 (ERK1/2), besides the activation of the SMA/MAD homologues protein (SMAD) pathway. Inhibition of the TGF-βtype 1 receptor, alias activin receptor-like kinase 5 (ALK5), hindered monocyte chemotaxis towards TGF-β1 and TGF-β1-activated downstream signalling cascades. Individual genetic knock-downs for receptor-regulated SMAD2 and SMAD3 did not affect monocyte migration to TGF-β1. Inhibition of phosphoinositide 3 kinase (PI3K) activity, but not AKT, diminished both basal and TGF-β1-mediated monocyte motility. TGF-β1-induced monocyte chemotaxis did not rely on ERK1/2, but rather on p38. Remarkably, TGF-β1 was able to stimulate chemotaxis of diabetic monocytes. The current study provides novel insights into the molecular mechanisms of TGF-β1-induced monocyte migration, requiring ALK5 kinase activity and signalling via PI3K and p38. TGF-β1-driven monocyte motogenicity is fully functional in diabetic conditions, which is in sharp contrast to the impaired chemotactic responses to certain other arteriogenic cytokines. Therefore, TGF-β1 may be a promising candidate for endogenously and exogenously stimulating collateral growth in diabetic patients.

Neurotrophic Tyrosine Kinase Receptor B/Neurotrophin 4 Signaling Axis Is Perturbed in Clinical and Experimental Pulmonary Fibrosis

The neurotrophins (NTs) are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in diseased lungs. In this study, the increased expression of NT4/5 and of its cognate receptor, the neurotrophic tyrosine kinase receptor Type 2 (TrkB), was observed in human lungs explanted from patients with idiopathic pulmonary fibrosis (IPF), and in lungs from mice with bleomycin-induced pulmonary fibrosis. The expression of NT4/5 and TrkB localized to hyperplastic alveolar Type II cells (ATII) and fibroblastic foci in affected lungs. Increased concentrations of NT4/5 and TrkB were evident in ATII isolated from the lungs of bleomycin-treated mice. Primary ATII were shown to secrete NT4/5 into the cell culture medium. The profibrotic cytokine transforming growth factor-β1, stimulated TrkB, but not NT4/5 gene expression, suggesting that perturbed profibrotic growth factor signaling in affected lungs may drive the expression of TrkB. NT4/5 enhanced the proliferation of ATII through a TrkB/extracellular-regulated kinase/protein kinase B pathway, and could also drive the proliferation of primary human and murine lung fibroblasts, through TrkB-dependent and protein kinase B-dependent pathways. Taken together, these data suggest that a dysregulated TrkB/NT4/5 axis may contribute to several of the pathological lesions associated with pulmonary fibrosis, including ATII hyperplasia and the proliferation of fibroblasts, and we would add IPF to the list of disorders, such as pain and cancer, for which therapeutic targeting of the TrkB/neurotrophin axis has been proposed for further investigation.

Divergent Contractile and Structural Responses of the Murine Protein Kinase C-ε Null Pulmonary Circulation to Chronic Hypoxia

Divergent Contractile and Structural Responses of the Murine Protein Kinase C-ε Null Pulmonary Circulation to Chronic Hypoxia

Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2α Protein Kinase GCN2*[boxs

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Protein Kinase C-associated Kinase (PKK), a Novel Membrane-associated, Ankyrin Repeat-containing Protein Kinase

A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

Isolation and Characterization of Two Evolutionarily Conserved Murine Kinases (Nek6 and Nek7) Related to the Fungal Mitotic Regulator, NIMA

Entrance and exit from mitosis in Aspergillus nidulans require activation and proteolysis, respectively, of the NIMA (never in mitosis, gene A) serine/threonine kinase. Four different NIMA-related kinases were reported in mammals (Nek1–4), but none of them has been shown to perform mitotic functions related to those demonstrated for NIMA. We describe here the isolation of two novel murine protein kinase genes, designated nek6 and nek7, which are highly similar to each other (87% amino acid identity in the predicted kinase domain). Interestingly, Nek6 and Nek7 are also highly similar to the F19H6.1 protein kinase of Caenorhabditis elegans (76 and 73% amino acid identity in the kinase domain, respectively), and phylogenetic analysis suggests that these three proteins constitute a novel subfamily within the NIMA family of serine/threonine kinases. In contrast to the other documented NIMA-related kinases, Nek6/7 and F19H6.1 harbor their catalytic domain in the C-terminus of the protein. Immunofluorescence suggests that Nek6 and Nek7 are cytoplasmic. Linkage analysis, using the murine BXD recombinant inbred strain panel, localized nek6 to chromosome 2 at 28 cM. Using a mouse/hamster radiation hybrid panel, we assigned the nek7 gene to chromosome 1 at approximately 73 cM.

A Novel Murine PKA-Related Protein Kinase Involved in Neuronal Differentiation

Members of the cAMP-dependent second-messenger pathway have been described as regulators of cellular growth and differentiation and were consequently implicated in a variety of embryogenic processes including brain development. Moreover, recent data suggest an indispensable role for cAMP-dependent protein kinases (PKAs) in neuronal differentiation and synaptic plasticity. Using a degenerate primer-based approach, we have identified a novel murine gene closely related to the human cAMP-dependent protein kinase PRKX on Xp22.3. This gene (Pkare) was mapped to the region near the centromere of the murine X chromosome and is expressed in a variety of adult organs including kidney, liver, spleen, testis, ovary, lung, heart, and brain. Antisense in situ hybridization on staged mouse embryos revealed a highly distinctive expression pattern during neuronal development, with elevated Pkare expression observed only in differentiating neurons within the first ganglion, the dorsal root ganglia, and the mantle layer of the telencephalon. Based on the close relationship with the catalytic PKA subunits and its distinct expression in differentiating neuronal cells, Pkare might represent a novel component of the cAMP-regulated pathways involved in brain development and function.

Cloning and Characterization of the Murine Nek3 Protein Kinase, a Novel Member of the NIMA Family of Putative Cell Cycle Regulators

We have cloned and characterized murine Nek3 (NIMA-related kinase 3), a novel mammalian gene product structurally related to the cell cycle-regulatory kinase NIMA of Aspergillus nidulans. By RNase protection, low levels of Nek3 expression could be detected in all organs examined, regardless of proliferative index. In contrast to Nek1 and Nek2, Nek3 levels were not particularly elevated in either the male or the female germ line. Nek3 levels showed at most marginal variations through the cell cycle, but they were elevated in G0-arrested, quiescent fibroblasts. Furthermore, no cell cycle-dependent changes in Nek3 activity could be detected, and no effects upon cell cycle progression could be observed upon antibody microinjection or overexpression of either wild-type or catalytically inactive Nek3. Finally, Nek3 was found to be a predominantly cytoplasmic enzyme. These data indicate that Nek3 differs from previously characterized Neks with regard to all parameters investigated, including organ specificity of expression, cell cycle dependence of expression and activity, and subcellular localization. Hence, the structural similarity between mammalian Neks may not necessarily be indicative of a common function, and it is possible that some members of this kinase family may perform functions that are not directly related to cell cycle control.

Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae.

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.

Carbon source‐dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECε) coding for the expression of murine protein kinase C epsilon (PKCε) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECε expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCε antibody. Extracts from these pYECε-transfected cells could phosphorylate a PKCε substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCε was fused in frame with GST (GST-Rε), further confirming the successful expression of murine PKCε. Induction of PKCε expression by galactose in cells transformed with pYECε increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCε expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCα or PKCβ-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCε expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCε expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCε-expressing yeast cells (1–2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCε expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCε expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCε-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells. J Cell Physiol 178:216–226, 1999. © 1999 Wiley-Liss, Inc.

MPK38 expression is upregulated in immature T cells activated by concanavalin A

We have previously identified a cDNA clone from a murine teratocarcinoma PCC4 cDNA library, which we have termed MPK38 for murine protein kinase 38. MPK38 is a new member of the SNF1 serine/threonine kinase family. To understand the role of the MPK38 in thymocytes, we have investigated the level of MPK38 expression by Northern blot analysis. Interestingly, incubation of thymocytes at 37°C resulted in the loss of the MPK38 transcript, however the transcript could be reinduced by treatment with Con A or PHA, but not with PMA and growth factors such as IL-2, IL-7, TGF- β, TNF- α, and EGF. In addition, stimulation of mature T cells from the spleen with Con A failed to reinduce the MPK38 transcript, indicating a developmental regulation of MPK38 expression, whereas non-T cell populations significantly reinduced the transcript. These results suggest that MPK38 may play a functional role as one of the signal regulators in early T cell activation and, potentially, certain lineages of hematopoietic cell activation induced by Con A.

Cloning, Expression Analysis, and Functional Characterization of PKL12, a Member of a New Subfamily of ser/thr Kinases

We report the cloning of the full-length cDNA of a new murine protein kinase, mPKL12. The sequence reveals a 305-amino-acid protein that contains the characteristic subdomains of the kinase superfamily and particular homology indicating a ser/thr specificity. We have also identified its human homologue gene (94% identical) and the putative homologue proteins fromSaccharomyces cerevisiaeandArabidoposis thaliana.These four sequences appear to form a new subfamily of protein kinases, close in size to the theoretical minimal catalytic domain, therefore suggesting that they could be the catalytic unit of a more complex holoenzyme. UsingEscherichia coli-purified protein, we have demonstrated that the mPKL12 enzyme possesses an intrinsic kinase activity, capable of phosphorylating enolase and also of promoting autophosphorylation, with a ser/thr specificity. Tissue expression analysis of mPKL12 showed that it is ubiquitously expressed, although at very low levels. RT-PCR analysis of several cell lines also supports this view, therefore suggesting that PKL12 may play a role in a very general cellular function, probably related with the secretory pathway.

Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus.

Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase, Nlk, that is a murine homolog of the Drosophila nemo (nmo) gene. The Nlk amino acid sequence is 54. 5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4. 0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60-70% of Nlk is localized to the nucleus, whereas 30-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.

Murine Protein Kinase CK2α′: cDNA and Genomic Cloning and Chromosomal Mapping

Protein kinase CK2 (casein kinase II) is a heterotetrameric enzyme implicated in many essential regulatory pathways in cells. We have determined the sequence of the murine CK2α′ cDNA that encodes a 350-amino-acid protein that would have 99 and 98% homology with the human and chicken proteins, respectively, and is also highly homologous to murine CK2α. To clarify the sequence of the 5′ end of the cDNA and to elucidate the structure and regulation of the gene, we obtained a bacterial artificial chromosome clone that contains the 35-kb CK2α′ gene. The gene consists of 12 small exons; the 5′ end, including the first exon and intron, is extremely GC rich and contains a CpG island. The putative promoter contains potential binding sites for a variety of transcriptional factors but appears to lack CCAAT- or TATA-like elements. A polymorphic dinucleotide repeat in the fifth intron allowed us to map the CK2α′ gene to murine Chromosome 8.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene.

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3' end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5' by approximately 2 kb revealing a 559-bp region showing strong homology to the proposed 5' untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of approximately 2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.