Direct Recruitment of ERK Cascade Components to Inducible Genes Is Regulated by Heterogeneous Nuclear Ribonucleoprotein (hnRNP) K
2011; Elsevier BV; Volume: 286; Issue: 11 Linguagem: Inglês
10.1074/jbc.m110.213330
ISSN1083-351X
AutoresMichał Mikula, Karol Bomsztyk,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoComponents of the ERK cascade are recruited to genes, but it remains unknown how they are regulated at these sites. The RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) K interacts with kinases and is found along genes including the mitogen-inducible early response gene EGR-1. Here, we used chromatin immunoprecipitations to study co-recruitment of hnRNP K and ERK cascade activity along the EGR-1 gene. These measurements revealed that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP K and RNA polymerase II (Pol II). Inhibition of EGR-1 transcription with either serum-responsive factor knockdown or 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole altered recruitment of all of the above ERK cascade components along this locus that mirrored the changes in Pol II and hnRNP K profiles. siRNA knockdown of hnRNP K decreased the levels of active MEK and ERK at the EGR-1, changes associated with decreased levels of elongating pre-mRNA and less efficient splicing. The hnRNP K dependence and pattern of ERK cascade activation at the c-MYC locus were different from at EGR-1. Ribonucleoprotein immunoprecipitations revealed that hnRNP K was associated with the EGR-1 but not c-MYC mRNAs. These data suggest a model where Pol II transcription-driven recruitment of hnRNP K along the EGR-1 locus compartmentalizes activation of the ERK cascade at these genes, events that regulate synthesis of mature mRNA. Components of the ERK cascade are recruited to genes, but it remains unknown how they are regulated at these sites. The RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) K interacts with kinases and is found along genes including the mitogen-inducible early response gene EGR-1. Here, we used chromatin immunoprecipitations to study co-recruitment of hnRNP K and ERK cascade activity along the EGR-1 gene. These measurements revealed that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP K and RNA polymerase II (Pol II). Inhibition of EGR-1 transcription with either serum-responsive factor knockdown or 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole altered recruitment of all of the above ERK cascade components along this locus that mirrored the changes in Pol II and hnRNP K profiles. siRNA knockdown of hnRNP K decreased the levels of active MEK and ERK at the EGR-1, changes associated with decreased levels of elongating pre-mRNA and less efficient splicing. The hnRNP K dependence and pattern of ERK cascade activation at the c-MYC locus were different from at EGR-1. Ribonucleoprotein immunoprecipitations revealed that hnRNP K was associated with the EGR-1 but not c-MYC mRNAs. These data suggest a model where Pol II transcription-driven recruitment of hnRNP K along the EGR-1 locus compartmentalizes activation of the ERK cascade at these genes, events that regulate synthesis of mature mRNA. IntroductionLocalized spatiotemporal activation of kinases is an important mode to regulate kinase specificity (1Shaul Y.D. Seger R. Biochim. Biophys. Acta. 2007; 1773: 1213-1226Crossref PubMed Scopus (687) Google Scholar). It has been known for more than 20 years that kinases can translocate to the nucleus (2Nigg E.A. Hilz H. Eppenberger H.M. Dutly F. EMBO J. 1985; 4: 2801-2806Crossref PubMed Scopus (197) Google Scholar, 3Seth A. Gonzalez F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. 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These observations are consistent with K protein acting as a docking platform within microcircuits where it serves to integrate signaling cascades by facilitating cross-talk between kinases and factors at sites of RNA-directed processes (22Adolph D. Flach N. Mueller K. Ostareck D.H. Ostareck-Lederer A. Mol. Cell. Biol. 2007; 27: 1758-1770Crossref PubMed Scopus (33) Google Scholar, 31Bomsztyk K. Denisenko O. Ostrowski J. BioEssays. 2004; 26: 629-638Crossref PubMed Scopus (373) Google Scholar, 32Van Seuningen I. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar).The extracellular signal-regulated kinases p44 (ERK1) and p42 (ERK2) belong the family of MAPKs (33Yao Z. Seger R. Biofactors. 2009; 35: 407-416Crossref PubMed Scopus (102) Google Scholar). They are activated by a variety of mitogenic ligands that bind to surface receptor tyrosine kinases (RTKs) (13McKay M.M. Morrison D.K. 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Nelson J.D. Castner D.G. Denisenko O. Bomsztyk K. Nucleic Acids Res. 2008; 36: e17Crossref PubMed Scopus (64) Google Scholar, 37Lawrence M.C. McGlynn K. Shao C. Duan L. Naziruddin B. Levy M.F. Cobb M.H. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13315-13320Crossref PubMed Scopus (55) Google Scholar). ERKs target a large repertoire of nuclear protein substrates; thus compartmentalization at genomic sites explains one way by which their specificity is controlled in the intracellular environment. Still, it is not known how they are recruited and regulated along genes.Early growth response-1 (EGR-1, two exons, 3,824-bp-long locus) is an immediate early gene that encodes a zinc finger transcription factor (38Sukhatme V.P. J. Am. Soc. Nephrol. 1990; 1: 859-866Crossref PubMed Google Scholar). EGR-1 is rapidly and transiently expressed in many cell types following mitogenic stimulation, which then targets genes involved in signal transduction and gene expression (39Kubosaki A. Tomaru Y. Tagami M. Arner E. Miura H. Suzuki T. Suzuki M. Suzuki H. Hayashizaki Y. Genome Biol. 2009; 10: R41Crossref PubMed Scopus (59) Google Scholar). hnRNP K is recruited to inducible gene loci including EGR-1. Intriguingly, ERK1/2 is bound to EGR-1 in a pattern that resembles K protein (36Flanagin S. Nelson J.D. Castner D.G. Denisenko O. Bomsztyk K. Nucleic Acids Res. 2008; 36: e17Crossref PubMed Scopus (64) Google Scholar). Moreover, hnRNP K binds (40Laury-Kleintop L.D. Tresini M. Hammond O. J. Cell. Biochem. 2005; 95: 1042-1056Crossref PubMed Scopus (22) Google Scholar), regulates (41Chang J.W. Koike T. Iwashima M. Int. Immunol. 2009; 21: 1351-1361Crossref PubMed Scopus (12) Google Scholar), and is a substrate (20Chen L.C. Liu H.P. Li H.P. Hsueh C. Yu J.S. Liang C.L. Chang Y.S. Oncogene. 2009; 28: 1904-1915Crossref PubMed Scopus (45) Google Scholar) of ERK1/2 and other components of the ERK cascade. These previously published studies may reflect compartmentalized hnRNP K-controlled bursts of ERK activity in the vicinity of inducibly transcribed genes. Here, we explored the role and interaction of hnRNP K with the ERK1/2 cascade along inducible genes following mitogenic stimulation.DISCUSSIONThe discovery that terminal kinases such as ERK have hundreds of protein substrates has fueled great interest to understand mechanisms that control their specificity (13McKay M.M. Morrison D.K. Oncogene. 2007; 26: 3113-3121Crossref PubMed Scopus (446) Google Scholar, 33Yao Z. Seger R. Biofactors. 2009; 35: 407-416Crossref PubMed Scopus (102) Google Scholar). Thus far, several elements have been identified that account for kinase specificity including amino acid consensus sequence, subcellular localization or compartmentalization, duration and magnitude of the signals, cross-talk with other pathways, and scaffolds that modulate their activity (1Shaul Y.D. Seger R. Biochim. Biophys. Acta. 2007; 1773: 1213-1226Crossref PubMed Scopus (687) Google Scholar, 12Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 13McKay M.M. Morrison D.K. Oncogene. 2007; 26: 3113-3121Crossref PubMed Scopus (446) Google Scholar). Below we discuss the elements of ERK specificity revealed in the current study and suggest a model for hnRNP-K-compartmentalized in situ control of ERK kinase cascade at the EGR-1 gene, which in turn regulates K protein-dependent mRNA synthesis and processing (Fig. 8).Compartmentalization of the ERK Cascade Signaling Module at Inducible GenesIt has been previously demonstrated that nearly all of the components of the ERK pathway translocate to the nucleus (73Oláh Z. Komoly S. Nagashima N. Joó F. Rapp U.R. Anderson W.B. Exp. Brain Res. 1991; 84: 403-410Crossref PubMed Scopus (26) Google Scholar, 74Romero F. Ramos-Morales F. Domínguez A. Rios R.M. Schweighoffer F. Tocqué B. Pintor-Toro J.A. Fischer S. Tortolero M. J. Biol. Chem. 1998; 273: 7776-7781Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 75Carpenter G. Curr. Opin. Cell Biol. 2003; 15: 143-148Crossref PubMed Scopus (142) Google Scholar, 76Lin S.Y. Makino K. Xia W. Matin A. Wen Y. Kwong K.Y. Bourguignon L. Hung M.C. Nat. Cell Biol. 2001; 3: 802-808Crossref PubMed Scopus (885) Google Scholar). This and another recent study from our laboratory (11Nelson J.D. LeBoeuf R.C. Bomsztyk K. Diabetes. 2011; 60: 127-137Crossref PubMed Scopus (33) Google Scholar) provide evidence that in fact most of the ERK cascade components, from GRB2 down to ERK, are inducibly recruited along an inducible gene (FIGURE 1, FIGURE 2, FIGURE 3). These signal transducers were not found in the intergenic regions flanking the EGR-1, indicating that the ERK activation cascade is compartmentalized to discrete sites, restricting their action to only relevant gene locus-associated substrates along the Pol II transcribed region.Timing of the ERK Cascade Induction along Inducible GenesUnder the conditions of the present study, the serum-induced levels of active MEK/ERK assessed by Western blot analysis in whole cell lysates (Fig. 4B) were relatively low. In sharp contrast, the induced ChIP levels of all the ERK cascade components at the EGR-1 and the Nr4A3 loci were robust and short-lived. Moreover, the temporal profiles of ERK cascade at EGR-1 and Nr4A3 were very different from the one at the c-MYC and hnRNP K (Fig. 5) or the GAPDH genes (11Nelson J.D. LeBoeuf R.C. Bomsztyk K. Diabetes. 2011; 60: 127-137Crossref PubMed Scopus (33) Google Scholar). The levels of active MEK/ERK seen by Western blot analysis represent the sum of MEK/ERK activities in different subcellular compartments/sites. Thus, the local burst of ERK activities occurring at different time points after mitogenic stimulation, as illustrated by the different profiles at EGR-1 and c-MYC (Fig. 5), would not be as readily detectable in bulk cell fractions.The short burst of localized activity (e.g. at the EGR-1 locus) would ensure that ERK will phosphorylate only those substrates that are present at the correct time in the immediate vicinity of the compartmentalized signaling cassette. The sharp peaks of activity will also provide a means for more precise cross-talk timing with other signaling cascades, further increasing specificity (52Kyriakis J.M. Biochim. Biophys. Acta. 2007; 1773: 1238-1247Crossref PubMed Scopus (26) Google Scholar). In the cases studied here, the activation of the ERK cascade components is linked to the elongating Pol II along inducible genes. The tight coupling to Pol II seems like an ideal way for these signal transducers to specifically regulate in a timely fashion serum-induced factors involved in co-transcriptional RNA processing (Fig. 8). In fact, a recent study suggests that synchronized activation of ERK cascade components along genes appears to be important in maintaining effective transcription (11Nelson J.D. LeBoeuf R.C. Bomsztyk K. Diabetes. 2011; 60: 127-137Crossref PubMed Scopus (33) Google Scholar).Activation of ERK at Inducible Gene LociThe unexpected kinetic differences between MEK-ERK activities measured in whole cell lysates compared with those measured at the specific genomic sites (Fig. 4) are not consistent with the traditional view of cytoplasmic ERK activation. Thus, these observations raise questions about the pathways that activate ERK at these genes in the absence of comparable changes in the cell as whole. Most of the ERK cascade components (77Zehorai E. Yao Z. Plotnikov A. Seger R. Mol. Cell. Endocrinol. 2010; 314: 213-220Crossref PubMed Scopus (89) Google Scholar) as well as RTKs (78Hsu S.C. Hung M.C. J. Biol. Chem. 2007; 282: 10432-10440Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 79Aleksic T. Chitnis M.M. Perestenko O.V. Gao S. Thomas P.H. Turner G.D. Protheroe A.S. Howarth M. Macaulay V.M. Cancer Res. 2010; 70: 6412-6419Crossref PubMed Scopus (160) Google Scholar, 80Podlecki D.A. Smith R.M. Kao M. Tsai P. Huecksteadt T. Brandenburg D. Lasher R.S. Jarett L. Olefsky J.M. J. Biol. Chem. 1987; 262: 3362-3368Abstract Full Text PDF PubMed Google Scholar) have previously been found in the nucleus, and mechanisms of their nuclear translocation are beginning to be defined (77Zehorai E. Yao Z. Plotnikov A. Seger R. Mol. Cell. Endocrinol. 2010; 314: 213-220Crossref PubMed Scopus (89) Google Scholar). These factors could be preassembled in the nucleus so that induction of EGR-1 and Nr4A3 may not require serum-induced nuclear translocation of these components. We have measured high constitutive levels of ERK components at the GAPDH gene (supplemental Fig. S2) (11Nelson J.D. LeBoeuf R.C. Bomsztyk K. Diabetes. 2011; 60: 127-137Crossref PubMed Scopus (33) Google Scholar). We have also found high constitutive levels of insulin receptor at insulin-responsive genes without detectable insulin (11Nelson J.D. LeBoeuf R.C. Bomsztyk K. Diabetes. 2011; 60: 127-137Crossref PubMed Scopus (33) Google Scholar). EGFR is also bound to genes and exhibits transcriptional activity (76Lin S.Y. Makino K. Xia W. Matin A. Wen Y. Kwong K.Y. Bourguignon L. Hung M.C. Nat. Cell Biol. 2001; 3: 802-808Crossref PubMed Scopus (885) Google Scholar). Thus, RTKs could also be constitutively preassembled in the nucleus and serve to initiate activation of the ERK cascade module at inducible gene loci. Because we did not detect RAS, Ran, known to be chromatin-associated and to interact with Shc (81George R. Chan H.L. Ahmed Z. Suen K.M. Stevens C.N. Levitt J.A. Suhling K. Timms J. Ladbury J.E. Cell Mol. Life Sci. 2009; 66: 711-720Crossref PubMed Scopus (10) Google Scholar), could be an alternative small GTPase that could link nuclear RTKs to Grb2/SOS. We have found that a specific MEK inhibitor, U0126, blocks inducible recruitment of all components, GRB2, SOS, B-Raf, MEK, and ERK, to the EGR-1 gene without any effects on histone marks (supplemental Fig. S5, rows 8 and 9). This observation suggests the presence of a different upstream activation ERK cascade module that indirectly controls the ERK signaling microcircuit at the EGR-1 gene. For example, a cytoplasmic ERK activation module could drive nuclear translocation, binding, and activation of SRF, Elk (Fig. 2 and supplemental Fig. S3), and/or other transcription factors to the EGR-1 locus initiating Pol II recruitment/elongation. In turn, hnRNP K binding activates the ERK microcircuit, a process that may involve all of the cascade components including chromatin-bound RTKs.hnRNP K acts as a docking platform at sites of RNA-directed processes (22Adolph D. Flach N. Mueller K. Ostareck D.H. Ostareck-Lederer A. Mol. Cell. Biol. 2007; 27: 1758-1770Crossref PubMed Scopus (33) Google Scholar, 31Bomsztyk K. Denisenko O. Ostrowski J. BioEssays. 2004; 26: 629-638Crossref PubMed Scopus (373) Google Scholar, 82Wolf D. Witte V. Clark P. Blume K. Lichtenheld M.G. Baur A.S. Cell Host Microbe. 2008; 4: 398-408Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). hnRNP K exists in complexes with ERK1/2 and regulates their activities (40Laury-Kleintop L.D. Tresini M. Hammond O. J. Cell. Biochem. 2005; 95: 1042-1056Crossref PubMed Scopus (22) Google Scholar, 82Wolf D. Witte V. Clark P. Blume K. Lichtenheld M.G. Baur A.S. Cell Host Microbe. 2008; 4: 398-408Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). We show that in response to serum treatment, hnRNP K recruitment along the EGR-1 gene is linked to Pol II transcription (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4). The hnRNP K knockdown inhibited MEK1/2 and ERK1/2 activities along the EGR-1 and NR4A3 genes (Fig. 5). This observation is consistent with a model where K protein is a functional scaffold for the ERK cascade along these loci.ERK Targets along Inducible GenesThere could be several targets of the K protein-mediated discrete activation and recruitment of ERK1/2 along the EGR-1 and NR4A3 genes. Inhibition of MEK-ERK activity by hnRNP K knockdown had little or no effect on Pol II occupancy along the EGR-1 and NR4A3 genes (Fig. 5), suggesting that processivity was not altered. Pol II processivity can be uncoupled from the rate of elongation (84Mason P.B. Struhl K. Mol. Cell. 2005; 17: 831-840Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Moreover, pre-mRNA processing is linked to Pol II elongation. Consistent with this view, we provide evidence that K protein regulates both the rate of elongation and splicing (Fig. 6). Thus, ERK cascade targets could include elongation and/or splicing factors.ERK1/2 phosphorylates Ser-284 and Ser-353 of hnRNP K (29Habelhah H. Shah K. Huang L. Ostareck-Lederer A. Burlingame A.L. Shokat K.M. Hentze M.W. Ronai Z. Nat. Cell Biol. 2001; 3: 325-330Crossref PubMed Scopus (246) Google Scholar). Thus, after hnRNP K-mediated ERK activation along the EGR-1 gene, the K protein itself may become ERK substrate regulating the splicing activity of K. ERK-mediated phosphorylation also drives K protein export from the nucleus (29Habelhah H. Shah K. Huang L. Ostareck-Lederer A. Burlingame A.L. Shokat K.M. Hentze M.W. Ronai Z. Nat. Cell Biol. 2001; 3: 325-330Crossref PubMed Scopus (246) Google Scholar). ERK-mediated phosphorylation of hnRNP K increases stability of K protein-bound transcripts (20Chen L.C. Liu H.P. Li H.P. Hsueh C. Yu J.S. Liang C.L. Chang Y.S. Oncogene. 2009; 28: 1904-1915Crossref PubMed Scopus (45) Google Scholar) and also drives K protein export from the nucleus (29Habelhah H. Shah K. Huang L. Ostareck-Lederer A. Burlingame A.L. Shokat K.M. Hentze M.W. Ronai Z. Nat. Cell Biol. 2001; 3: 325-330Crossref PubMed Scopus (246) Google Scholar). Thus, the ERK-catalyzed phosphorylation can also play a role in hnRNP K-bound EGR-1 mRNA stability and transport to the cytoplasm (Fig. 8).In sum, we obtained evidence that nearly the full complement of the ERK cascade components is recruited along the inducible genes coupled to Pol II elongation. Binding of hnRNP K that accompanies these signal transducers plays a role in MEK1/2 recruitment and ERK1/2 activation along the inducible EGR-1 and NR4A3 loci. This study illustrates for the first time how a spatiotemporally restricted chain of events starting with Pol II recruitment followed by K protein binding to inducible loci, presumably to nascent transcript, controls kinases, which in turn regulate rate of mRNA synthesis and processing. In this regard, compartmentalized intracellular bursts of activation of kinase signaling cascades at different time points following mitogenic activation could be a more ubiquitous phenomenon than previously recognized. IntroductionLocalized spatiotemporal activation of kinases is an important mode to regulate kinase specificity (1Shaul Y.D. Seger R. Biochim. Biophys. Acta. 2007; 1773: 1213-1226Crossref PubMed Scopus (687) Google Scholar). It has been known for more than 20 years that kinases can translocate to the nucleus (2Nigg E.A. Hilz H. Eppenberger H.M. Dutly F. EMBO J. 1985; 4: 2801-2806Crossref PubMed Scopus (197) Google Scholar, 3Seth A. Gonzalez F.A. Gupta S. Raden D.L. Davis R.J. J. Biol. 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These previously published studies may reflect compartmentalized hnRNP K-controlled bursts of ERK activity in the vicinity of inducibly transcribed genes. Here, we explored the role and interaction of hnRNP K with the ERK1/2 cascade along inducible genes following mitogenic stimulation.
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