Cloning And Characteristics Of People Hssad1 Analysis Cum Hssad1 Of Protein Kinase Function For 14-3-3 Regulation Mechanism

Protein kinases mediate most of the signal transduction in eukaryotic cells; by modification of substrate activity, protein kinases also control many other cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation.Based on homologous screening strategy, we cloned a novel human BRSK kinase family member HsSAD1 gene. The HsSAD1 cDNA is 3109bp in length, encoding a protein of 778 amino acid residues, mapped to human chromosome 19q13.4 and composed of 19 exons. The 34-285 amino acid residues of amino acid of HsSAD1 consist of a serine/threonine protein kinase domain. The in vitro kinase assay showd that wild-type HsSAD1 has the kinase activity, while its K63M mutant lost the kinase activity. The kinase activity of HsSAD1 contributes its proper localization. The wild-type HsSAD1 distributes both in nucleus and cytoplasm in HEK293 cells, while catalytically inactive HsSAD1 localized only in cytoplasm. Northern blot analysis revealed that HsSAD1 transcript was expression with significant highly in brain and testis. There also was expressed in heart, prostate, thymus, uterus and pancreas. The expression patterns of human brain tissue membrane showed that HsSAD1 was ubiquitously expressed in cerebellum, cerebral cortex, medulla, occipital pole, frontal lobe, temporal lobe and putamen, but low levels expression in spinal cord. Human brain cDNA library was screened through two yeast hybridization, which using inactive HsSAD1 as bait. RanBPM, MAP1Band 14-3-30 were found to interact with HsSAD1 in yeast.For the BRSK kinases are not constitutively active kinases, but the kinases regulated by other interaction proteins. Based on 14-3-3β was a partner protein ofHsSAD1 in yeast and as known 14-3-3 proteins were interacting protein of many kinases, so we identified that 14-3-3 protein was a regulatory protein of HsSAD1 by in vivo and in vitro binding assays. Members of 14-3-3 protein family are highly conserved and functions are some difference. They consist of seven isoforms inhuman( η,β,γ,σ,τ,ε,ζ). To investigate whether if binding of HsSAD1 and 14-3-3 proteins is isoform-specific interaction, we cloned all the human 14-3-3isoforms form brain cDNA library except 14-3-3β and confrmed their interactionwith HsSAD1. The results indicated that among the seven 14-3-3 isoforms tested only 14-3-3η,β,γ,σ could bind to HsSAD1 in cells. Members of the 14-3-3 family of proteins typically interact with phosphoprotiens. To elucidate HsSAD1 was a phosphoprotien when interacted with 14-3-3 β protein, we replaced Lys-51 , Arg- 58 and Lys-129 with alanine in 14-3-3 β protein( 14-3-3 β^K51AR58A and 14-3-3 β^R129A), which the residues mutations exposed on the binding surface impair the phosphopeptide binding ability. No interaction between 14-3-3 β^K51AR58A or 14.3.3 β^R129A with HsSAD1 respectably was detected, indicated that HsSAD1 may be recognized by 14-3-3 β as a phosphoprotein. While dephosphorylated HsSAD1 with λ -PPase also lost its binding ability with GST 14-3-3 β. We found that catalytically inactive HsSAD1 reduced its binding with 14-3-3 β and wild type HsSAD1 has a ability of autophosphrylation. These findings demonstrate a requirement of HsSAD1autophosphrylation for efficient 14-3-3β binding. To determine which motif wasresponsible for the binding, we design a set of deletions to identify the interaction motif in HsSAD1. The binding assay results showed the most possible motif locates in HsSAD1 349-500 aa. To find the role of HsSAD1-14-3-3β complex formation on HsSAD1 kinase activity, we found that the over-expression of 14-3-3 could reduce HsSAD1 kinase activity in cells. Moreover, incubation with recombinant 14-3-3β decreased HsSAD1 kinase activity. These results indicated that 14-3-3 is not only a HsSAD1 associating protein but also a protein that negatively regulates HsSAD1 kinase activity. During the cell cycle assays, we also found that 14-3-3β protein could partly reverse HsSAD1-inducible G2/M arrest in 293T cells. This data suggest that 14-3-3 protein was the important regulatory of HsSAD1 in cells.Moreover, here we report the cloning and characterization of another novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase member AGPAT7 gene. 1-acyl-sn-glycerol-3-phosphate acyltransferase is crucial enzyme for synthesis ofglycerolipids as well as triacylglylcerol biosynthesis in eukaryotes. Six members of 1-acyl-sn-glycerol-3-phosphate acyltransferase family in human have been described, which were AGPAT1, 2, 3, 4, 5 and 6. The AGPAT7 was mapped to human chromosome 15q14. The AGPAT7 cDNA is 1898 bp in length, encoding a putative protein with 524 amino acid residues, which contains an acyltransferase domain in 123 - 234 aa. RT PCR amplification in eighteen human tissues indicated that human AGPAT7 gene was widely expressed in uterus, thymus, pancreas, skeletal muscle, bladder, stomach, lung and testis. AGPAT7 protein was mainly localized to the endoplasmic reticulum (ER) in Hela cells.