Schmidt Lab:Notebook/Transcriptome Analysis

From OpenWetWare

<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Schmidt Lab:Notebook/Transcriptome Analysis</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>

Owwnotebook icon.png <sitesearch>title=Search this Project</sitesearch>

Customize your entry pages Help.png

Project Description/Abstract

Analysis of transcriptome data from Heat Stress Project


Protein Kinase:Substrate List
LMH cells 2.5 hr heat stress.

iGEP Used iGEP to identify kinases in complete gene list from LMH cells -included genes not differentially regulated.

Identified a total of 245 kinases in the LMH expression data. Expression of these 217 did not respond to heat stress, 19 were down-regulated and 9 were up-regulated.

Identified all kinases that were either up or down regulated.

PRKCB and PRK2B were the two kinases differentially regulated in both CELC and LMH.

PRKCB Involved in modulation of glucose response.For example: Inhibition of PKCbeta improves glucocorticoid-induced insulin resistance in rat adipocytes . PMID 12665248

PTK2B This gene encodes a cytoplasmic protein tyrosine kinase which is involved in calcium-induced regulation of ion channels and activation of the map kinase signaling pathway. The encoded protein may represent an important signaling intermediate between neuropeptide-activated receptors or neurotransmitters that increase calcium flux and the downstream signals that regulate neuronal activity. The encoded protein undergoes rapid tyrosine phosphorylation and activation in response to increases in the intracellular calcium concentration, nicotinic acetylcholine receptor activation, membrane depolarization, or protein kinase C activation. This protein has been shown to bind CRK-associated substrate, nephrocystin, GTPase regulator associated with FAK, and the SH2 domain of GRB2. The encoded protein is a member of the FAK subfamily of protein tyrosine kinases but lacks significant sequence similarity to kinases from other subfamilies.

PLK1 was down-regulated and had the greatest number of substrates. Used WebGIVI to determine iTerms for PLK1 and its substrates. From Uniprot: "Polo-like kinase Serine/threonine-protein kinase that performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, the removal of cohesins from chromosome arms, the inactivation of anaphase-promoting complex/cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis. "
Downregulation of PLK1 by RNAi leads to reduction in cell proliferation and increased incidence of apoptosis in transfected human cancer cell lines. SW-480 cells exhibited mitotic arrest with loss of centrosomal function.
PMID 18829454Protein kinase D ( PKD ) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol .

MKNK2 Map kinase interacting serine/threonine kinase 2"Eukaryotic initiation factor 4E (eIF4E) controls a crucial step of translation initiation and is critical for cell growth . Biochemical studies have shown that it undergoes a regulated phosphorylation by the MAP-kinase signal-integrating kinases Mnk1 and Mnk2 . Although the role of eIF4E phosphorylation in mammalian cells has remained elusive , recent work in Drosophila has established that it is required for growth and development . Here, we demonstrate that a previously identified Drosophila kinase called Lk6 is the functional homolog of mammalian Mnk kinases. We generated lk6 loss-of-function alleles and found that eIF4E phosphorylation is dramatically reduced in lk6 mutants. Importantly, lk6 mutants exhibit reduced viability, slower development, and reduced adult size, demonstrating that Lk6 function is required for organismal growth."
Will up-regulation of MKNK2 promote survival by maintaining translation of necessary transcripts?

SIK1 Salt Inducible Kinase"SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes,"
Salt-inducible kinase-1 represses cAMP response element-bindingprotein activity both in the nucleus and in the cytoplasm
Starvation Response To examine the role of SIK in vivo, we generated Drosophila SIK loss-of-function mutants and found that the mutant flies have higher amounts of lipid and glycogen stores and are resistant to starvation."
So will up-regulation of SIK promote glucose utilization?


(To insert image go to upload file page, upload the image then replace example with image title.)

Green are down regulated genes while orange are expressed but unchanged by heat stress.
TPT1 modulates HSP90
NUDC To explore the underlying mechanism , we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90 , which is also associated with LIS1 . Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, which are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites.
SUGT1 To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones.
NPM1 Nucleophosmin Nuclear chaperone.

Other Substrates
Wee1Phosphorylation of Wee1A by these kinases (PLK1 and CDC2) cooperatively stimulated the recognition and ubiquitination of Wee1A by SCF(beta-TrCP1/2) in vitro.
CDC20 activates the anaphase-promoting complex and essential for transition from metaphase to anaphase. Phosphorylation by PLK1 promotes degradation of CDC20 during G1 phase but did not affect ability of CDC20 to form APC checkpoint complexes.
Uniprot_BUB1B "Essential component of the mitotic checkpoint. Required for normal mitosis progression. The mitotic checkpoint delays anaphase until all chromosomes are properly attached to the mitotic spindle". Phosphorylation (by PLK1) does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate.
TOPORS Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser(718) in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation.