Sarah Rice - US grants

DESCRIPTION (provided by applicant): The mechanism by which truncated kinesin dimers hydrolyze ATP and move unidirectionally along microtubules is well understood. It is far less clear how the full kinesin heterotetramer, which has two heavy chains and two light chains, is regulated and activated for cargo transport. In this work, we will test the hypothesis that kinesin is regulated when the tails directly bind the heads to prevent ADP release or microtubule binding. Kinesin may be further regulated by a charge clash between its light chains and microtubules, and kinesin may be re-activated when phosphorylated light chains compete the tails away from the heads. We will test these hypotheses in four Specific Aims. The first two Aims address regulation using the full-length kinesin heavy chain, and the second two Aims explore the role of the light chains in regulation and activation. In Aim #1, we will determine whether the tail binds directly in the microtuble-binding site or to the nucleotide-sensing elements in the head, or whether it allosterically affects the nucleotide- or microtubule-binding regions of the head. The experiments of Aim #2, guided by the results of Aim #1, will determine what region of the head binds the tail and will identify specific head-tail interactions. Experiments performed both in vivo and in vitro indicate that the light chains may have a significant role in regulating kinesin, which will be assessed in Aim #3. Lastly, we will determine whether phosphorylation of kinesin light chains can directly activate kinesin in Aim #4. Together, these experiments will extend our understanding of the interactions and conformational changes that govern kinesin activity. Furthermore, the regulatory interactions that are found in this work may reveal inhibitory mechanisms that are similar in several kinesins. This may lead to quicker discovery of drugs that specifically target kinesins.

The molecular motor kinesin-1 performs a large number of transport tasks, and the regulatory mechanisms governing those processes are critical. Mis-regulation of kinesin-1 or mis-localization of kinesin-1 cargoes may be implicated in several diseases such as Parkinson¿s disease, neurofibromatosis, schizophrenia, and Charcot-Marie-Tooth disease. Kinesin-1¿s motile mechanism is well understood, and we now also know that kinesin-1¿s C-terminal tail interacts directly with and inhibits the heads when the motor is not needed for cargo transport. However, we do not know how kinesin-1 regulators initiate or stop cargo movement. The tail is certainly involved, as it binds to heads, microtubules, and several distinct kinesin-1 activators that function in different transport complexes. Separate from the tail, the Miro protein has a direct, Ca2+-dependent interaction with kinesin-1¿s enzymatic head domains, and Miro is required for Ca2+-dependent suppression of mitochondrial motility. We hypothesize that the tail is an intrinsically disordered domain, having structural flexibility that facilitates multiple binding partner interactions involved in kinesin-1 auto-inhibition and/or activation, while Miro has a distinct mechanism, directly inhibiting the enzymatic mechanism of kinesin-1 heads to suppress mitochondrial movement. To address this hypothesis, we will first gain detailed information in vitro about the structure of the kinesin-1 tail and its interactions with binding partners, by NMR and EPR spectroscopy. We will determine whether Miro is a direct, Ca2+-switchable inhibitor of kinesin-1¿s enzymatic activity, assess its effects on kinesin-1 mechanism using EPR, and map its interaction with kinesin-1 heads by cross-linking. After obtaining this structural and mechanistic information on both the tails and Miro, we will determine whether and how they influence mitochondrial movement by controlling kinesin-1 in vivo, by imaging mitochondria in live Drosophila S2 cells. These Aims together will provide an exciting new bridge between in vitro biophysical and cell biological work on molecular motor transport mechanisms. Furthermore, as Miro and other kinesin-1 regulators have been implicated in several neurological diseases, our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.

DESCRIPTION (provided by applicant): This proposal is based on a new finding that Src family kinases (SFKs) phosphorylate human Eg5, an essential mitotic kinesin family motor protein. SFKs are the original, canonical oncogenes and Eg5 is critical for spindle pole separation and stabilization of the mitotic spindle. Both proteins are anti-mitotic drug targets, with inhibitors in Phase I and II trials. Our preliminary data indicates that Src phosphorylates th enzymatic Eg5 heads at three tyrosines in vitro and in cells. These tyrosines are structurally very near the binding sites for Eg5 inhibitors. Our preliminary data also show that phosphomimetic mutations inhibit Eg5 activity and block the binding of an Eg5 inhibitor, STLC. We hypothesize that SFK phosphorylation of Eg5 heads alters Eg5 activity, localization, and action in bipolar spindle assembly and maintenance. We further hypothesize that SFK phosphorylation blocks the binding of several Eg5-targeted inhibitors. To begin our study, we will determine the mechanistic effects of SFK phosphorylation in vitro. We will develop functional phosphomimetic mutants and phospho-specific antibodies for the SFK sites in Eg5 heads. Next, we will examine the effects of SFK phosphoregulation of Eg5 on the progression of mitosis in fixed and live LLC-PK1 cells. We will develop new methods and cell lines for imaging the effects of SFK phosphorylation on mitotic targets, which will enable researchers to address the neglected issue of SFK activity in mitosis for any target of choice. The final aim of this study isto test whether SFK phosphorylation directly affects Eg5 inhibitor binding and efficacy in vitro and in cells. These experiments will be a first step in evaluating a potential combination therapy regimen targeting SFKs and Eg5.