Browsing by Subject "Kinesin"
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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.Item Regulation of the Cytoskeleton by Kinesins(2013-11-20) Weil, Lauren Melissa; Albanesi, Joseph P.; Cobb, Melanie H.; Conrad, Nicholas; Scherer, PhilippKinesins are motor proteins that associate with microtubules. The position of the motor domain has been linked to kinesin function. While amino-terminal and carboxy-terminal localization of the motor domain is linked to cargo transport, kinesins with the motor domain in the middle (M-kinesins) have a role in microtubule depolymerization. The kinesin-13 family consists of four M-kinesins, KIF2A, KIF2B, KIF2C, and KIF24. These proteins regulate the cytoskeleton through their microtubule depolymerizing activity. All four kinesins have reported functions in mitosis, while little is known about their roles in interphase. KIF2A and KIF2C are upregulated in cancer cells and the increased protein expression influences cell migration and invasiveness. In order to understand how KIF2A and KIF2C influence migration, we analyzed the microtubule and actin cytoskeleton in cells manipulated for kinesin expression. We found that depletion of KIF2A increases the number of focal adhesions and stress fibers and results in defects in cell spreading. KIF2A does not influence the dynamics of focal adhesion assembly or disassembly. In contrast, depletion of KIF2C prevents re-formation of focal adhesions. has little or no effect on the actin cytoskeleton. Here we uncovered a functional divergence in regulation of the cytoskeleton between KIF2A and KIF2C. Furthermore, this is the first time that an M-kinesin, which does not transport cargo, has been shown to influence focal adhesion dynamics.Item Structural Basis for Coordination in Dimeric Kinesin(2009-09-04) Metlagel, Zoltan; Kikkawa, MasahideKinesin-1 (conventional kinesin) is a protein motor that carries organelles and vesicle cargo along its microtubule track. The two catalytic heads of Kinesin-1 are linked to function as a highly processive "molecular walker'' that can take hundreds of steps before falling off the track. A key requirement for processivity is that the nucleotide cycles of the heads are coordinated to prevent simultaneous release of both heads from the track. The structural basis for coordination has not been established yet. Here, we show the conformational changes involved in nucleotide-dependent switching of the kinesin core in the functional context of the \MT. The observed conformational differences between two key nucleotide states comprise the structural groundwork for future studies on how the nucleotide cycles are coordinated between the heads. Further, a software suite, Ruby-Helix, was developed to facilitate helical image analysis and implement a new algorithm for the analysis of helical objects with a seam. Ruby-Helix incorporates several new techniques for conventional helical analysis, and automates many of the repetitive steps involved in helical analysis, thereby greatly increasing the throughput of this method.