John W. Tullai

Immediate-early and delayed primary response genes are distinct in function and genomic architecture

The transcriptional program induced by growth factor stimulation is classically described in two stages as follows: the rapid protein synthesis-independent induction of immediate-early genes, followed by the subsequent protein synthesis-dependent induction of secondary response genes. In this study, we obtained a comprehensive view of this transcriptional program. As expected, we identified both rapid and delayed gene inductions. Surprisingly, however, a large fraction of genes induced with delayed kinetics did not require protein synthesis and therefore represented delayed primary rather than secondary response genes. Of 133 genes induced within 4 h of growth factor stimulation, 49 (37%) were immediateearly genes, 58 (44%) were delayed primary response genes, and 26 (19%) were secondary response genes. Comparison of immediateearly and delayed primary response genes revealed functional and regulatory differences. Whereas many immediate-early genes encoded transcription factors, transcriptional regulators were not prevalent among the delayed primary response genes. The lag in induction of delayed primary response compared with immediateearly mRNAs was because of delays in both transcription initiation and subsequent stages of elongation and processing. Consistent with increased abundance of RNA polymerase II at their promoters, immediate-early genes were characterized by over-representation of transcription factor binding sites and high affinity TATA boxes. Immediate-early genes also had short primary transcripts with few exons, whereas delayed primary response genes more closely resembled other genes in the genome. These findings suggest that genomic features of immediate-early genes, in contrast to the delayed primary response genes, are selected for rapid induction, consistent with their regulatory functions.

Thiol Activation of Endopeptidase EC 3.4.24.15 A NOVEL MECHANISM FOR THE REGULATION OF CATALYTIC ACTIVITY

Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show that both recombinant and tissue-derived EP24.15 are activated up to 8-fold by low concentrations (0.1 mm) of dithiothreitol. Additionally, under non-reducing conditions, recombinant and native EP24.15 forms multimers that can be returned to the monomeric form by reduction. We have also shown that competitive inhibitor binding occurs only to the monomeric form, which indicates that catalytic site access is restricted in the multimeric forms. Through systematic site-directed mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant (C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24.15 activity is regulated through changes in intra- and extracellular redox potential.

Glycogen Synthase Kinase-3 Represses Cyclic AMP Response Element-binding Protein (CREB)-targeted Immediate Early Genes in Quiescent Cells

Despite its central role in cell survival and proliferation, the transcriptional program controlled by GSK-3 is poorly understood. We have employed a systems level approach to characterize gene regulation downstream of PI 3-kinase/Akt/GSK-3 signaling in response to growth factor stimulation of quiescent cells. Of 31 immediate-early genes whose induction was dependent on PI 3-kinase signaling, 12 were induced directly by inhibition of GSK-3. Most of the GSK-3-regulated genes encoded transcription factors, growth factors, and signaling molecules. Binding sites for CREB were highly over-represented in the upstream regions of these genes, with 9 genes containing CREB sites that were conserved in mouse orthologs. Binding sites predicted in 6 genes were confirmed by CREB chromatin immunoprecipitation and forskolin induction of CBP binding. Moreover, CREB siRNA substantially blocked induction of 5 genes by forskolin and of 3 genes following inhibition of GSK-3. These results indicate that GSK-3 actively represses gene expression in quiescent cells, with inhibition of CREB playing a key role in this transcriptional response.

Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15).

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.

The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation

The metalloendopeptidase EC 3.4.24.15(EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 ± 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH1–9, bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K m andk cat for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.

GSK-3 Represses Growth Factor-inducible Genes by Inhibiting NF-κB in Quiescent Cells

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-κB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IκB kinase, leading to phosphorylation and degradation of IκBα and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-κB through inhibition of IκB kinase. This inhibition of NF-κB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.

A GSK-3-mediated transcriptional network maintains repression of immediate early genes in quiescent cells

Glycogen synthase kinase-3 (GSK-3) plays a central role in cell survival and proliferation, in part by the regulation of transcription. Unlike most protein kinases, GSK-3 is active in quiescent cells in the absence of growth factor signaling. In a recent series of studies, we employed a systems-level approach to understanding the transcription network regulated by GSK-3 in a quiescent cell model. We identified a group of immediate early genes that were upregulated in quiescent cells solely by the inhibition of GSK-3 in the absence of growth factor stimulation. Computational analysis of the upstream sequences of these genes identified statistically over-represented binding sites for the transcription factors CREB, NFκB and AP-1, and the roles of these factors in regulating expression of GSK-3 target genes were verified by chromatin immunoprecipitation and RNA interference. In quiescent cells, GSK-3 inhibits CREB, NFκB and AP-1, thereby maintaining repression of their target genes and contributing to maintenance of cell cycle arrest.

Regulated expression during development and following sciatic nerve injury of mRNAs encoding the receptor tyrosine phosphatase HPTPζ/RPTPβ

Three major isoforms of the receptor protein tyrosine phosphatase HPTPzeta/RPTPbeta (RPTPzeta/beta) have been previously identified, two with identical transmembrane and intracellular catalytic domains that differ by virtue of a long cysteine-free extracellular region, and a soluble proteoglycan called phosphacan that lacks the transmembrane and carboxy-terminal catalytic domains. To determine whether these RPTPzeta/beta variants are produced by alternative mRNA splicing of a common primary transcript, we performed genomic Southern analysis and characterized several rat cDNA and genomic RPTPzeta/beta clones. These studies indicated that the three major transcripts which encode phosphacan and the two RPTPzeta/beta phosphatase variants are encoded by a single gene, and further that additional alternative mRNA splicing is likely to result in the deletion of a 7 amino acid insert from the intracellular juxtamembrane region of both long and short phosphatase isoforms. Simultaneous quantitation of the three major isoforms by RNase protection analysis indicated that the mRNA encoding phosphacan had the highest relative abundance in the CNS while that encoding the short phosphatase isoform was most abundant relative to the other RPTPzeta/beta variants in the PNS. Following peripheral nerve crush, all RPTPzeta/beta mRNAs, including phosphacan and the phosphatase variants with and without the 21 base insert, were significantly induced in the distal segments of the sciatic nerve with a time course that correlated well with the response of Schwann cells to this injury.