KINESIN PHOSPHORYLATION

  • Scholey, Jonathan M, (PI)

Project: Research project

Project Details

Description

The ATPase, kinesin, which transports particles towards the plus-ends of
microtubules (MTs) in vitro, is thought to participate in a variety of
important cellular and developmental processes including mitosis, nuclear
migrations, vesicle trafficking, and organizing intracellular membranes.
The structural and functional characterization of kinesin is progressing
rapidly, but little is known about the regulation of kinesin activity. We
previously purified and characterized kinesin from sea urchin eggs and
embryos, and found that it consists of 2 mol 130Kd heavy chain (HC) : 1 mol
84Kd light chain (LC) : 1 mol 78Kd LC. Recently we have observed that the
HC and LCs can be phosphorylated in vivo and in vitro, and here we propose
a detailed characterization of these phosphorylation reactions, together
with a study of their effects on kinesin-driven intracellular transport. We will use phosphoamino acid analysis, phosphopeptide mapping and
radiosequencing methods to determine the number, location and sequence of
the phosphorylation sites on kinesin HC and LC. Phosphopeptide antibodies
will be raised for use in monitoring the abundance and distribution of
phosphorylated kinesin in cells and subcellular fractions. We will use
kinesin or kinesin peptides as substrates for characterizing kinesin
phosphorylation-dephosphorylation reactions, in assays for purifying
kinesin kinases and phosphatases from sea urchin cytosol, and comparing
them to previously-characterized enzymes (a number of which will also be
screened for kinesin kinase or phosphatase activity). If this work reveals
that a kinesin kinase or phosphatase is a novel enzyme we will characterize
its localization, as well as its structural and biochemical properties. To
investigate the effects of phosphorylation on the intracellular
distribution of kinesin, subcellular fractionation and immunocytochemistry
will be performed under conditions that enhance or inhibit kinesin
phosphorylation. Most important, we will compare the activities of
phosphorylated and nonphosphorylated kinesin preparations using motility
assays, membrane binding/transport assays, and MT-activated ATPase assays.
Thus we hope to learn if HC and/or LC phosphorylation controls (1) the
mechanochemical activity of kinesin and (2) the attachment of kinesin to
its transported cargo.
StatusFinished
Effective start/end date7/1/916/30/96

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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