Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117

We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117.     

Isolation and characterization of mouse testis specific serine/threonine kinase 5 possessing four alternatively spliced variants

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5alpha, Tssk5beta, Tssk5gamma and Tssk5delta. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5alpha which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5alpha, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5alpha exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5beta, TSSK5gamma and TSSK5delta cannot be stimulated at the CREB/CRE responsive pathway in cmparison to TSSK5alpha. These findings suggest that TSSK5beta, TSSK5gamma, TSSK5delta may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5beta, TSSK5gamma and TSSK5delta can directly interact with TSSK5alpha. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.     

A novel serine/threonine kinase gene,Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. © 1996 Wiley-Liss, Inc.     

Tssk1和Tssk2的克隆、表达及其在成熟精子中的分布与亚细胞定位

睾丸特异性丝氨酸/苏氨酸蛋白激酶(Testis-SpecificSerine/Threonine Kinases,TSSKs)可能在精子发生和(或)精子功能调节中起着重要作用,该文研究克隆并表达了小鼠Tssk1和Tssk2基因,纯化得到了Tssk1和Tssk2蛋白激酶,经Western blotting分析,Tssk1和Tssk2皆存在于小鼠和人的成熟精子中。免疫组化的结果显示,Tssk1分布于小鼠的头部(顶体)及整个尾部,Tssk2主要分布在小鼠精子头部顶体后的区域;获能前后的小鼠精子其Tssk1及Tssk2分布模式未发生改变,小鼠Tssk2在诱发顶体反应前后精子中的分布模式也无变化。然而,原来存在于顶体的Tssk1在诱发顶体反应后由于顶体的丢失而未能检出其信号,但尾部的信号不受影响。在人精子中,Tssk1分布区域为颈部及尾部,Tssk2则分布于赤道板的位置。研究结果提示,Tssk1和Tssk2可能对精子功能具有重要调节作用。     

The Structure,Role, and Regulation of Type 1 Protein Phosphatases

Abstract

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase F_A/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, α- and β-adrenergic agonists, glucocorticoids, and thyroid hormones.     

Identification of a novel human MAST4 gene, a new member of human microtubule associated serine/threonine kinase family

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here, we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64, 63, 59, and 39% identical amino acid residues with MAST1, MAST2, MAST3, and MASTL, respectively. RT-PCR analysis revealed a relatively high expression level of MAST4 in most normal human tissues, with the exception of in testis, small intestine, colon, and peripheral blood leukocyte. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 808–815. The text was submitted by the authors in English. The nucleotide sequences reported in this paper have been submitted to GenBank under accession number: AY830839. These two authors contributed equally to this paper.     

Molecular cloning, characterization and expression of PmRsr1, a Ras-related gene from yeast form of Penicillium marneffei

GTP-binding proteins such as Ras act as molecular switches in a large number of signal pathways. In this report, we isolated and characterized a novel Ras small monomeric GTPase Rsr1 gene, designated PmRsr1, from yeast-form Penicillium marneffei. The full-length PmRsr1 cDNA sequence is 1,866 bp in size, and contains an open reading frame of 642 bp encoding 213 amino acids. The predicted molecular mass of PmRsr1 is 24.41 kDa with an estimated theoretical isoelectric point of 9.21. The deduced amino acid sequence of PmRsr1 shows 87% identity with that of Aspergillus fumigatus and A. clavatus. Eight exons and seven introns are identified within the 2,102 bp PmRsr1 genomic DNA sequence of P. marneffei. The open reading frame was subcloned into the pcDNA6-myc-His B expression vector, and the recombinant plasmid was transfected into Vero cell line. The expressed fusion protein was analyzed by SDS-PAGE and western blotting. Differential expression of the PmRsr1 was demonstrated by real-time RT-PCR. The expression of PmRsr1 was the highest in the yeast phase comparing with that in the mycelia and conidia phases.     

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full‐length complementary (c)DNA of interleukin‐1 receptor‐associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine–threonine‐specific protein kinase catalytic domain and a C‐terminal domain. The amino‐acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84·5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up‐regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up‐regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.     

Isolation and characterization of <Emphasis Type="Italic">NgRLK1</Emphasis>, a receptor-like kinase of <Emphasis Type="Italic">Nicotiana glutinosa</Emphasis> that interacts with the elicitin of <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (~10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn²⁺ than Mg²⁺.     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus,a developmental bacterium exhibiting social behavior

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like protein serine/threonine kinases in the prokaryotes. © 1993 Wiley-Liss, Inc.     

Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities

Aplog-1 is a simplified analog of the tumor-promoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of aplog-1. In this study, we synthesized amide-aplog-1 (3), in which the C-1 ester group was replaced with an amide group, to improve chemical stability in vivo. Unfortunately, 3 exhibited seventy-fold weaker binding affinity to the C1B domain of PKCδ than that of aplog-1, and negligible anti-proliferative and cytotoxic activities even at 10^?4 M. A conformational analysis and density functional theory calculations indicated that the stable conformation of 3 differed from that of aplog-1. Since 27-methyl and 27-methoxy derivatives (1, 2) without the ability to bind to PKC isozymes exhibited marked anti-proliferative and cytotoxic activities at 10^?4 M, 3 may be an inactive control to identify the target proteins of aplogs.     

Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.     

Two novel protein kinase genes,OsMSRPK1 andOsMSURPK2, are regulated by diverse environmental stresses in rice

Two novel rice (Oryza sativa L.) protein kinase (PK) genes have been isolated.OsMSRPK1 andOsMSURPK2, which most likely exist as single-copy genes in the rice genome, encode 693 and S03 amino acids polypeptide, respectively, and have the serine/threonine kinase domain of cyclin dependent protein kinase (OsMSRPK1), or the serine/threonine kinase domain and NAF domain (OsMSURPK2). Steady-state mRNA analyses of these PKs, with constitutive expression in the leaves of two-week-old seedlings, revealed thatOsMSRPK1 is up-regulated upon exposure to environmental stresses, whereasOsMSVRPK2 is down-regulated by these same stresses. Furthermore, the two PKs are developmentally regulated in both young and mature rice plants, including in the panicles. These results strongly suggest that the genes have roles in both plant development and in their defense/stress-signaling pathways.