Browsing by Subject "Mitogen-Activated Protein Kinase 3"
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Item Identification and Characterization of the Multifunctional Epigenetic Regulator CFP1 as an ERK1/2 Substrate(2014-11-21) Klein, Aileen Melanie; Sternweis, Paul C.; Cobb, Melanie H.; Goodman, Joel M.; Conrad, NicholasEpigenetic regulation of gene transcription occurs as an integration of multiple layers of signals at a genetic locus. These signals can include local chromatin structure, covalent modifications to both histone proteins and DNA, the presence of transcription factors, and modification directly to the transcriptional machinery. Our lab is interested in the control of cellular processes by the mitogen activated protein kinases ERK1/2. In a yeast two-hybrid screen with activated ERK2 (extracellular signal-regulated kinase 2) to find novel interacting partners, our lab identified CFP1 (CxxC finger protein 1), a DNA-binding protein that is a vital component of the H3K4 trimethylating Set1A/B complexes to promote gene transcription. CFP1 has also been shown to interact physically and functionally with the major maintenance DNA methyltransferase DNMT1. We are interested in defining how substrate targeting of CFP1 by ERK1/2 regulates downstream transcriptional outcomes. Interaction between ERK2 and CFP1 in cells was validated by co-immunoprecipitation from isolated mononucleosomes. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro, an observation supported by studies in cells. Some of the most likely in vivo ERK1/2 phosphorylation sites include serine 224 and threonine 227. CFP1 is essential for focusing trimethylation of H3K4 at promoters, a histone modification that supports transcription from these loci. We hypothesized that phosphorylation of CFP1 by ERK1/2 during mitogenic signaling may support trimethylation of H3K4 and transcription of ERK1/2-regulated target genes. Introduction of CFP1 containing the mutation T227V into HeLa cells blocked global H3K4 trimethylation to a similar extent as CFP1 depletion. On the other hand, CFP1 S224A shows diminished transactivation capacity against a model transcriptional substrate. Neither of these mutants fail to interact with Set1B in a pulldown, suggesting that these sites may be important for Set1 complex targeting or activity towards chromatin. Consistently, CFP1 knockdown hinders induction of several ERK1/2-regulated immediate early gene targets in response to serum treatment. It will be of interest to test whether this is dependent on stable or inducible H3K4 trimethylation and what impact overexpression of point mutants will play in their transcription. Regulation of H3K4 trimethylation through CFP1 phosphorylation might represent a novel regulatory input to support transcription of ERK1/2-regulated genes.Item Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein(2009-06-17) Jivan, Arif; Cobb, Melanie H.Initially identified in Chlamydomonas, radial spoke protein 3 (RSP3) is one of at least twenty identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be identified and fully characterized. Mammalian RSP3 was found to interact with ERK2 through a yeast two-hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2. Confirming this finding, the human homolog long form, RSP3H, co-immunoprecipitates with ERK1/2 in HEK293 cells. Human RSP3, and its larger alternative start site gene product, radial spoke protein 3 homolog (RSP3H), are phosphorylated by ERK1/2 on threonine 286 in vitro and in cells. RSP3/RSP3H are also phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Additionally, we showed that human RSP3H functions as an A-kinase anchoring protein (AKAP), and its ability to bind to the regulatory subunits of PKA, RII and RII, is regulated by ERK1/2 activity and phosphorylation. Interestingly, expression analysis of mRNA suggests RSP3/RSP3H are also present in cells that are thought to contain a single primary cilium but not motile cilia. Immunofluorescence staining of primary cilia-containing cells indicates that RSP3/RSP3H localize to nuclear punctae, specifically promyelocytic leukemia (PML) bodies, suggesting a non-cilia related role for RSP3/RSP3H in these cells. Functionally, RSP3/RSP3H may localize ERK1/2 to a distinct site of action within the cell and serve as a point of convergence of cAMP-dependent and PKA-mediated influence upon ERK1/2 downstream signaling or vice versa. These data are the first to establish a connection between ERK1/2 and what was once ostensibly thought to only be a ciliary component as well as to identify a novel ERK1/2-interacting AKAP.