Browsing by Subject "Extracellular Signal-Regulated MAP Kinases"
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Item Functional Significance of Extracellular Signal Regulated Kinase (ERK2) Phosphorylation States: Implications for DNA Binding(2013-11-04) McReynolds, Andrea Christine; Rice, Luke M.; Cobb, Melanie H.; Albanesi, Joseph P.; Yu, GangThe protein kinase extracellular signal-regulated kinase 2 (ERK2) has been well understood structurally for nearly twenty years. New insight is emerging about its structure and function. A novel autophosphorylation has recently been found to occur on a critical active site residue, threonine 188. Autophosphorylation of this residue has been suggested to occur as a result of the confluence of receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) signaling pathways. The possibility of autophosphorylation on threonine 188 seemed to be inconsistent with what was known about the structure and function of protein kinases generally and ERK2 in particular; T188 and the comparable residue in other protein kinases is required for catalytic activity. I found ERK2 phosphorylated on T188 in vitro in partially purified preparations of the recombinant protein purified following expression in bacteria. This suggests that phosphorylation of ERK2 on T188 can occur without the input of upstream RTK or GPCR signaling. Through mutagenesis experiments, I found mutation of T188 sharply reduced activity toward substrates in vitro. Protein fractions containing pT188 and purified ERK2 T188D and T188E mutants, that may act as phosphomimetics, appear to have an increased affinity for DNA binding. I examined critical residues in the activation loop important for phosphorylation and activation of ERK2 and found that perturbation of these residues influences DNA binding specificity. Although the MAPK pathway and role of ERK2 is well understood, our data suggest that previously unrecognized, higher-order signaling mechanisms that arise from additional phosphorylation events may be involved in less well characterized properties of ERK2. Second, our finding that T188 autophosphorylation can occur in recombinant ERK2 independently from upstream GPCR signaling illustrates the need for a reinvestigation of the regulation of ERK2 autophosphorylation. Finally, direct and specific DNA binding may be driven by differences in phosphorylation. The result of this work serves to redefine a most important signaling molecule in terms of its structural modifications and relationship to overall function.Item Kinetics and Regulation of Protein Kinases(2015-06-23) Piala, Alexander Townshend; Rosenbaum, Daniel M.; Goldsmith, Elizabeth J.; Phillips, Margaret A.; De Brabander, Jef K.One of the major functions of kinases in biological systems is the relay of signal from an effector to a downstream target. Kinases are regulated by a diversity of mechanisms, both internal and external to the kinase. When the regulation of these kinases is somehow abrogated, disease can result. As such, understanding of these regulatory mechanisms could serve as useful in not only understanding cellular signal propagation, but also the disease states that can arise from their malfunction. Taking advantage well-developed projects in the Goldsmith lab, three systems were studied: two kinases and one kinase cascade. The MAP3K Thousand and One Kinase 2 was the subject of an inhibitor discovery program using high-throughput screening of a large small-molecule library. Secondly, the mechanism by which the kinase With No Lysine(K) 1 responds to chloride concentration in order to modulate downstream signaling was studied. Finally, a cascade comprising the MAP3K Apoptosis Signaling Kinase 1, MAP/ERK Kinase 6, and p38a was studied through modeling approaches using data derived from mass spectrometry. In each case, mechanisms for regulation were discovered. Tight-binding inhibitors of TAO2 were found that may prove useful in both in in vitro signaling studies as well as future in vivo work. WNK1 was shown to be regulated by direct chloride binding, which inhibits its auto-phosphorylation and thusly its auto-activation. Finally, important kinetic parameters for MAPK cascade signaling were determined, and it was demonstrated that additional species outside the cascade do not appear necessary for switch-like behavior under physiological-like in vitro conditions. These diverse regulatory mechanisms showcase how protein kinases manage to synthesize diverse inputs into physiologically meaningful downstream signals.Item Regulation by ERK1/2 of Novel Substrates, Kinesins KIF2A and KIF2C(2013-04-15) Zaganjor, Elma 1981-; Albanesi, Joseph P.; Cobb, Melanie H.; White, Michael A.; Fontoura, BeatrizThe kinesin-like protein KIF2A is a microtubule-associated motor protein thatauses microtubule depolymerization by inducing a conformational change in tubulin. The depolymerase function of KIF2A is utilized in mitotic cells as it is required to establish proper, bipolar spindles. Studies in KIF2A knockout mice revealed KIF2A function in regulation of interphase microtubules as KIF2A-/- neurons exhibit abnormal axon branching. Though protein kinases are known to regulate the mitotic function of KIF2A, how KIF2A is regulated in interphase cells has not been studied. In a yeast-two hybrid screen, designed to preferentially uncover interactors of the active form of ERK2, we identified KIF2A. We find that, human KIF2A can be v phosphorylated in vitro by pERK1/2 and that the kinases interact with KIF2A in cells. Through phosphorylation prediction tools and mutagenesis we identified threonine 78 (T78) as a major pERK2 phosphorylation site on KIF2A. Inhibition of ERK1/2 prevents KIF2A from localizing at the leading edge of cells. Additionally, knockdown of KIF2A phenocopies the effect of inhibiting ERK1/2 on microtubules; both treatments result in elongated microtubules. These data suggest that ERK1/2 may regulate KIF2A localization which in turn may be important for KIF2A function in depolymerizing microtubules. The close relative, KIF2C can also bind to and be phosphorylated by pERK1/2 in vitro, but the functional significance of this event remains unknown. In a laboratory generated non-small cell lung cancer (NSCLC) in which oncogenic K-RasG12V has been overexpressed and the tumor suppressor p53 has been knocked down, we found an increase in expression of KIF2A and KIF2C. This increase could be suppressed by inhibiton of the effector pathway RAS-RAF-MEK1 but not the PI3K pathway. As it is accepted that cancer cells have more dynamic microtubules that give them a migratory advantage, we hypothesized that upregulation of KIF2A and KIF2C also promote migration. Indeed, knockdown of KIF2A and KIF2C resulted in reduced migration in cancer cell lines. Microarray studies that had been performed on lung cancer lines revealed upregulation of KIF2A and KIF2C in cancers, suggesting that these proteins may be significant factors in the development of lung cancer. Finally, KIF2A and KIF2C regulate lysosomal dynamics. This regulation has an impact on signaling, particularly for mTORC1 which requires lysosomal localization for its activity.