Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases

Protein kinases frequently play key roles in the normal regulation of growth and development in eukaryotic organisms. As a consequence, aberrant expression or mutations in this family of molecules frequently result in transformation. Previously, we have conducted a screen to identify protein kinases that are expressed in the mouse during mammary gland development and in breast cancer cell lines. We now describe the molecular cloning, characterization and expression of Krct, a novel serine/threonine protein kinase unrelated to previously defined families of protein kinases. At the mRNA level, Krct is widely expressed throughout murine development and in adult tissues. Despite its ubiquitous expression, Krct is expressed preferentially within specific cellular compartments in multiple tissues, in particular within the testis and gastrointestinal tract. At the amino acid level, Krct is most closely related to four previously undescribed kinases in Saccharomyces cerevisiae, Arabidopsis thaliana and Caenorhabditis elegans. Together, these kinases appear to define a novel subfamily of serine/threonine protein kinases. Krct possesses an unusually long 5'-untranslated region containing multiple upstream initiation codons and, in this regard, is similar to many proto-oncogenes that regulate normal growth and differentiation. In addition, Krct is located on mouse chromosome 11 closely linked to the epidermal growth factor receptor and, therefore, is likely to be co-amplified in a variety of human tumors.     

The lipid phosphatase activity of PTEN is critical for its tumor supressor function

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.     

What's new in ras genes? Physiological role of ras genes in signal transduction and significance of ras gene activation in tumorigenesis

Ras gene mutations have been found with variable prevalence in different tumor types. While during the past decade a lot of information has been accumulated on the frequency of ras oncogene activation in tumors, the last two years brought considerable progress in elucidating molecular mechanisms of signal transduction for which cellular ras proteins are key elements. They transmit signals from upstream tyrosine kinases to downstream serine/threonine kinases ultimately leading to changes of gene expression cytoskeletal architecture, cell-to-cell interactions and metabolism. These signalling pathways are of interest for the physiological regulation of proliferation and differentiation in normal, as well as in cancer tissue. Mutational activation of cellular ras genes to transforming oncogenes is thought to promote cell growth even in the absence of extracellular stimuli, and may thereby contribute to the initiation and/or progression of tumors.     

Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S-->A) or mimicking (S-->D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

ZAP-70 and defects of T-cell receptor signaling

The activation, function, and development of peripheral T lymphocytes are dependent on the ability to signal properly through the surface T-cell antigen receptor (TCR)-CD3 complex. Transmission of such signals requires the activation of specific cytoplasmic protein tyrosine kinases (PTK) associated with the TCR. Recently, mutations in one such PTK, called ZAP-70, have been shown to be responsible for a rare, autosomal recessive form of severe combined immunodeficiency syndrome (SCID) in humans. This distinctive SCID syndrome is characterized by the selective absence of peripheral CD8+ T cells and by the presence of circulating CD4+ T cells that do not respond to TCR-mediated stimuli in vitro. T-cell immunodeficiency syndromes that arise as a consequence of inherited mutations in either the CD3epsilon or CD3gamma subunit proteins have also been described in rare patients. Absence of these TCR components results in severely decreased expression of the surface TCR-CD3 complex and defective signal transduction through the TCR. In this report, the clinical, laboratory, and molecular findings of these immunodeficiency disorders are described, insights are provided by these inherited defects into the pathways of TCR signal transduction, and T-cell development is discussed.     

Mutational analysis of N-RAS and GAP-related domain of the neurofibromatosis type 1 gene in chronic myelogenous leukemia

RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.     

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors

The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-specific manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.     

Src64 is required for ovarian ring canal morphogenesis during Drosophila oogenesis

The Src family of protein tyrosine kinases have been implicated as important regulators of cellular proliferation, differentiation and function. In order to understand further the role of Src family kinases, we have generated loss-of-function mutations in Src64, one of two Src family kinases known in Drosophila melanogaster. Animals with reduced Src64 function develop normally and are fully viable. However, Src64 female flies have reduced fertility, which is associated with the incomplete transfer of cytoplasm from nurse cells to the developing oocyte. Analysis of Src64 egg chambers showed defects in the ring canals that interconnect the oocyte and its 15 associated nurse cells. Src64 ring canals fail to accumulate the high levels of tyrosine phosphorylation that are normally present. Despite the reduced tyrosine phosphorylation, known ring canal components such as filamentous actin, a ring canal-specific product of the hu-li tai shao gene, and the kelch protein localize properly. However, Src64 ring canals are reduced in size and frequently degenerate. These results indicate that Src64 is required for the proper growth and stability of the ovarian ring canals.     

Identification of 21 novel human protein kinases, including 3 members of a family related to the cell cycle regulator nimA of Aspergillus nidulans

The nimA gene encodes a protein-serine/threonine kinase that is required along with the p34cdc2 kinase for mitosis in Aspergillus nidulans. We have searched for human protein kinases that are related to the NIMA protein kinase using the polymerase chain reaction. Different pairs of degenerate oligonucleotides specific for conserved amino acid motifs in the catalytic domain of NIMA were used as primers in the polymerase chain reaction to amplify partial complementary DNAs (cDNAs) of protein kinases expressed in the promyelocytic leukemia cell line HL-60. Forty-one distinct cDNAs representing a broad spectrum of serine/threonine- and tyrosine-specific protein kinases were identified, and the sequences for 21 of these protein kinases were found to be unique. Three of these cDNAs represent a family of protein kinases whose members are related to NIMA and the murine nimA-related protein kinase Nek1. We discuss the success of this polymerase chain reaction approach with respect to the use of multiple primer pairs, the influence of primer degeneracy, and the tolerance of cDNA amplification to mismatches between primers and template mRNA.     

Phosphatidylinositol 4-kinases

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. The first committed step is catalysed by phosphatidylinositol 4-kinase leading to the formation of phosphatidylinositol 4-phosphate. In the last four years, ten cDNA molecules have been cloned which code isoforms of phosphatidylinositol 4-kinase; some of which are highly related. Characteristically, they contain a C-terminal catalytic domain which is similar to that of (poly)phosphoinositide 3-kinases and to that of more distantly related lipid/protein kinases. Alignment has characterised cDNAs from Chaenorabditis, Dictyostelium and Schizostaphyloccus pombe as those of phosphatidylinositol 4-kinases also. All these lipid kinases are related to the superfamily of protein kinases. Several amino acids are highly conserved in catalytic domains of lipid and protein kinases. Employing the catalytic subunit of the cAMP-dependent protein kinase as template, these residues can be assigned functionally. On the basis of the alignment, a phylogenetic tree of the superfamily of phosphatidylinositol kinases has been constructed. Three families, the phosphatidylinositol 4-kinases, phosphoinositide 3-kinases, and the phosphatidylinositol related lipid/protein kinases, can be recognised. Each family comprises two subfamilies. The involvement of the phosphatidylinositol 4-kinases in signal transduction processes is summarised and a new hypothesis for the function of their isoforms in polyphosphoinositide signalling is presented. The involvement of phosphatidylinositol 4-kinases in formation of lipid-protein interactions with cytoskeleton proteins and the metabolism of polyphosphoinositide in the nucleus is discussed.     

Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms

The majority of familial Alzheimer's disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N-terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its approximately 20 kDa CTF involving protein kinase C (PKC) and/or protein kinase A (PKA). We now have found that the CTF of the highly homologous PS-2 is also phosphorylated. Surprisingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, the PS-2 CTF is constitutively phosphorylated in vivo by serine/threonine protein kinases, which are independent of phorbol ester and intracellular cAMP. In vitro the large hydrophilic loop of PS-2 between transmembrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK-1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed that the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosphorylate the CTF of PS-2 within its hydrophilic loop domain in vivo. Interestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.     

Mutations in the human skeletal muscle chloride channel gene (CLCN1) associated with dominant and recessive myotonia congenita

Myotonia, defined as delayed relaxation of muscle after contraction, is seen in a group of genetic disorders that includes autosomal dominant myotonia congenita (Thomsen's disease) and autosomal recessive myotonia congenita (Becker's disease). Both disorders are characterized electrophysiologically by increased excitability of muscle fibers, reflected in clinical myotonia. These diseases are similar except that transient weakness is seen in patients with Becker's, but not Thomsen's disease. Becker's and Thomsen's diseases are caused by mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1). Genetic screening of a panel of 18 consecutive myotonia congenita (MC) probands for mutation in CLCN1 revealed that a novel Gln-68-Stop nonsense mutation predicts premature truncation of the chloride channel protein. Four previously reported mutations, Arg-894-stop, Arg-338-Gln, Gly-230-Glu, and del 1437-1450, were also noted in our sample set. The Arg-338-Gln and Gly-230-Glu mutations were found in patients with different phenotypes from those of previous reports. Further study of the Arg-338-Gln and Gln-230-Glu alleles may shed light on variable modes of transmission (dominant versus recessive) in different families. Physiologic study of these mutations may lead to better understanding of the pathophysiology of myotonia in these patients and of voltage-gated chloride channel structure/function relationships in skeletal muscles.     

Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction

The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.