Isolation and characterization of a serine/threonine protein kinase SOS2 gene from Brassica napus

A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants.     

Expression cloning of a variety of novel protein kinases in Lotus japonicus

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the lambdaZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants.     

KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine.

cDNAs encoding KEPI, a novel protein kinase C (PKC)-potentiated inhibitory protein for type 1 Ser/Thr protein phosphatase (PP1), were identified. They were found among morphine-regulated brain mRNAs identified using subtracted differential display techniques. Full-length rat, mouse, and human cDNA and genomic sequences were elucidated with library screening and data base searching. Rat, mouse, and human KEPI cDNAs encode 164-165 amino acid proteins with calculated isoelectric points of 5.2. Each species' amino acid sequence contains consensus sequences for phosphorylation by PKC (KVT(72)VK), protein kinase A (RKLS(154)), and casein kinase II (S(43)SRE, S(120)EEE). Multiple KEPI N-terminal myristoylation consensus sites provide potential regions for membrane anchoring. Subcellular fractionation and Western analyses revealed that most KEPI immunoreactivity was associated with P2 and P3 membrane-enriched fractions and little in cytosolic fractions. 2.6-kb KEPI mRNAs were detected in brain, especially in the cerebral cortex and hippocampus, and in heart and skeletal muscle. Brain KEPI mRNA was up-regulated by both acute and chronic morphine treatments. The human KEPI gene contains four exons extending over more than 100 kb of genomic sequence on 6q24-q25, near the mu opiate receptor gene. These sequences displayed sufficient homology with the porcine PP1 inhibitor CPI-17 that we asked whether KEPI could share the ability of CPI-17 to modulate PP1 activity in a PKC-dependent fashion. Recombinant mouse KEPI is phosphorylated by PKC with a K(m) of 2.6 microm and a t(1/2) of 20 min. Phospho-KEPI inhibits PP1alpha with an IC(50) of 2.7 nm, a potency more than 600-fold greater than that displayed by unphosphorylated KEPI. Neither phospho- nor dephospho-KEPI inhibits protein phosphatase 2A. Up-regulation of KEPI expression by morphine, an agonist at PKC-regulating G-protein-coupled mu receptors, provides a novel signaling paradigm in which the half-lives of serine/threonine phosphorylation events can be influenced by activities at G(i)/G(o)-coupled receptors that modulate KEPI expression, KEPI phosphorylation, and KEPI regulation of PP1 activity.     

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.     

Up-regulation of a thioredoxin peroxidase-like protein, proliferation-associated gene, in hibernating bats

Two-dimensional gel electrophoresis was used to assess differential protein expression between euthermic and hibernating states in heart of Myotis lucifugus. A hibernation-induced protein was identified by mass spectrometry as a thioredoxin peroxidase-like protein known as PAG. Western blotting confirmed up-regulation (>2-fold) and RT-PCR also revealed up-regulation (>5-fold) of pag mRNA. Cloning revealed a highly conserved sequence suggesting a conserved function for PAG. Oxidative stress markers, p-IkappaB-alpha (Ser 32) and p-HSP27 (Ser 78/82), were also up-regulated in heart and skeletal muscle during hibernation. Although there are selected increases in gene/protein expression during hibernation, general translation inhibition occurs as part of metabolic rate depression. This was confirmed by elevated levels of the inactive forms of the eIF2alpha (Ser 51) in both heart and skeletal muscle (2- to 5-fold higher than in euthermia) and the eEF2 (Thr 51) in skeletal muscle (a 15-fold increase). This study suggests that hibernators may use up-regulation of specific proteins to counteract oxidative stress.     

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.     

Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine.

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.     

Molecular Cloning of a Gene on Chromosome 19q12 Coding for a Novel Intracellular Protein: Analysis of Expression in Human and Mouse Tissues and in Human Tumor Cells, Particularly Reed–Sternberg Cells in Hodgkin Disease

A novel protein, named NNX3, was molecularly characterized by cloning its cDNA, and its gene was mapped to chromosome 19q12. The equivalent mouse cDNA and gene were also cloned to allow us to analyze expression in murine in addition to human cells and tissues. Human and mouse NNX3 genes are composed of nine exons coding for proteins that are unrelated to any known protein. Signal peptides and hydrophobic domains are absent, corroborating their localization in the cytoplasm in transfected Cos cells. In Western blotting and immunoprecipitation, human NNX3 appeared as a doublet ofM_r64K–66K, while mouse NNX3 was a 70-kDa protein, both apparently much larger than the predicted 50 kDa, due in part to a stretch of 16–18 acidic residues hinging two nearly equally sized domains. In addition, phosphorylation of serine residues was demonstrated. Putative nuclear targeting signals were predicted, but NNX3 protein and two truncated versions remained localized in the cytoplasm of transfected Cos cells. NNX3 was expressed in embryonic and adult mouse tissues, particularly in brain, muscle, and lung. The expression of human NNX3 was most notable in human skeletal muscle and in ganglion cells and was also evident in human tumors and derived cell lines. This was confirmed by entries appearing in the GenBank EST database during the later phase of this study, representing partial NNX3 cDNA isolated from diverse neoplastic and developing tissues. Surprisingly, NNX3 was immunochemically detected in Reed–Sternberg cells of Hodgkin disease, in parallel with restin, a cytoplasmic protein we previously characterized (J. Delabieet al.,1993,Leuk. Lymphoma 12,21–26). The cloning and comprehensive molecular analysis of NNX3 as presented will form the basis for elucidating its function and, conversely, will constitute a marker for Reed–Sternberg cells in Hodgkin disease.     

Phosphorylation of adipose triglyceride lipase Ser404 is not related to 5'-AMPK activation during moderate-intensity exercise in humans

Intramyocellular triacylglycerol provides fatty acid substrate for ATP generation in contracting muscle. The protein adipose triglyceride lipase (ATGL) is a key regulator of triacylglycerol lipolysis and whole body energy metabolism at rest and during exercise, and ATGL activity is reported to be enhanced by 5'-AMP-activated protein kinase (AMPK)-mediated phosphorylation at Ser(406) in mice. This is a curious observation, because AMPK activation reduces lipolysis in several cell types. We investigated whether the phosphorylation of ATGL Ser(404) (corresponding to murine Ser(406)) was increased during exercise in human skeletal muscle and with pharmacological AMPK activation in myotubes in vitro. In human experiments, skeletal muscle and venous blood samples were obtained from recreationally active male subjects before and at 5 and 60 min during exercise. ATGL Ser(404) phosphorylation was not increased from rest during exercise, but ATGL Ser(404) phosphorylation correlated with myosin heavy chain 1 expression, suggesting a possible fiber type dependency. ATGL Ser(404) phosphorylation was not related to increases in AMPK activity, and immunoprecipitation experiments indicated no interaction between AMPK and ATGL. Rather, ATGL Ser(404) phosphorylation was associated with protein kinase A (PKA) signaling. ATGL Ser(406) phosphorylation in C(2)C(12) myotubes was unaffected by 5-aminoimidazole-4-carboxaminde-1-β-d-ribofuranoside, an AMPK activator, and the PKA activator forskolin. Our results demonstrate that ATGL Ser(404) phosphorylation is not increased in mixed skeletal muscle during moderate-intensity exercise and that AMPK does not appear to be an activating kinase for ATGL Ser(404/406) in skeletal muscle.     

Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells

Phosphorylation plays an important role in regulation of protein kinase C delta (PKCdelta). To date, three Ser/Thr residues (Thr 505, Ser 643, and Ser 662) and nine tyrosine residues (Tyr 52, Tyr 64, Tyr 155, Tyr 187, Tyr 311, Tyr 332, Tyr 512, Tyr 523, and Tyr 565) have been defined as regulatory phosphorylation sites for this protein (rat PKCdelta numbering). We combined doxycycline-regulated inducible gene expression technology with a hypothesis-driven mass spectrometry approach to study PKCdelta phosphorylation pattern in colorectal cancer cells. We report identification of five novel Ser/Thr phosphorylation sites: Thr 50, Thr 141, Ser 304, Thr 451, and Ser 506 (human PKCdelta numbering) following overexpression of PKCdelta in HCT116 human colon carcinoma cells grown in standard tissue culture conditions. Identification of potential novel phosphorylation sites will affect further functional studies of this protein, and may introduce additional complexity to PKCdelta signaling.     

From mosquito to man: Identification of a novel protein kinase, HsHPK, which is highly expressed in human hepatoma tissues

Protein kinases play an important role in the signaling pathway of growth factors in most of the higher organisms. During the study of protein kinase profiles of mosquitoes using RT-PCR and degenerate primers for consensus catalytic domain motifs to amplify protein kinase genes, we have noticed that a novel mosquito kinase, AaPK-38, shares a stretch of amino acids identical to the corresponding domain in Tousled gene ofArabidopsis thaliana that is required for leaf and flower development. A 2.1-kb cDNA encoding human HsHPK gene, which is a homolog of AaPK-38, was isolated from human testis cDNA library. This cDNA contains an open reading frame of 563 amino acids, with a complete kinase domain in its carboxyl terminus. The expressed Flag-tagged HsHPK was shown to have kinase activity based on in vitro autophosphorylation. Northern blot analysis revealed that human HsHPK mRNA is most abundant in testes, much less in heart and skeletal muscle and almost undetectable in liver and lung. Finally, we found that the expression of HsHPK in 4 out of 6 human hepatoma tissues is much higher than that in the adjacent normal counterpart. This result suggests HsHPK may play a role in the development of human hepatoma.     

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly sug     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.     

Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120.

Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.     

cDNA sequence, tissue-specific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3' untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12----qter region of chromosome 1. The locus is designated TNNI1.