A critical role for Kalirin in NGF signaling through TrkA

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction

Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates

Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood.Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly.PK genomic architecture, the size of gene functional domains and evolutionary rates correlate with the pattern of gene expression. Structure and evolutionary divergence of tissue-specific PK genes is related to the proliferative activity of the tissue where these genes are predominantly expressed. Our data provide evidence that physiological requirements for transcription intensity, ubiquitous expression, and tissue-specific regulation shape gene structure and affect rates of evolution

Data from: TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis

Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX Ser139 phosphorylation (γH2AX), and replacement of histones by protamines. Previously, we reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in nuclei of developing mouse spermatids. RNA-Seq analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6 KO sperm contained increased histones H3 and H4, and protamine 2 precursor/intermediate(s) indicative of a defective histone to protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility

Distinct Domains in the SHP-2 Phosphatase Differentially Regulate Epidermal Growth Factor Receptor/NF-κB Activation through Gab1 in Glioblastoma Cells

The transcription factor nuclear factor κB (NF-κB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-κB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-κB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-κB activation. We also show that EGFR-induced NF-κB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-κB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-κB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-κB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-κB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-κB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-κB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-κB activation

RNA-Seq data of WT and Tssk6 KO spermatids

Spermatids were purified from three sets of adult wild type (WT) and Tssk6 knockout (KO), total RNA was isolated, RNA-Seq libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kit, and sequenced on Illumina HiSeq 2500. Quality of raw data was assessed and passed by FastQC and reads were mapped to the reference genome (Mus musculus_UCSC_mm10). Cufflinks (v2.2.1) was used to estimate transcript levels represented by FPKM (fragments per kilo bases per million mapped reads) and differential expression between samples was determined by Cuffdiff (v.2.2.1). Student’s t-test was performed to calculate the P values

Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction

Tssk6 is required for Izumo relocalization and gamete fusion in the mouse

One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization