Sarah E. Rice, Ph.D. - 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.

Acute; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Amentia; Binding Proteins; Biophysical Process; Body Tissues; CRISP; Characteristics; Circumscribed Lobar Atrophy of the Brain; Computer Retrieval of Information on Scientific Projects Database; Corticodentatonigral degeneration with neuronal achromasia; Data; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Development; Disease; Disorder; FTD with parkinsonism; FTD-parkinsonism linked to chromosome 17; FTDP-17; Filament; Funding; Grant; Human; Human, General; Institution; Investigators; Ligand Binding Protein; Lobar Atrophy (Brain); MT-bound tau; Man (Taxonomy); Man, Modern; Micro-tubule; Microtubules; Morphology; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neurons; Numbers; Pathology; Pattern; Pick Disease of the Brain; Pick's Disease; Primary Senile Degenerative Dementia; Progressive Supranuclear Ophthalmoplegia; Progressive Supranuclear Palsy; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Solutions; Source; Steele-Richardson-Olszewski Disease; Steele-Richardson-Olszewski Syndrome; Structure; Supranuclear Palsy, Progressive; Tauopathies; Therapeutic Agents; Tissues; United States National Institutes of Health; abnormally aggregated tau protein; aggregation of microtubule associated protein tau; aggregation of microtubule-associated protein tau; base; brain tissue; chromosome 17-linked FTD with parkinsonism; cortico-basal degeneration; corticobasal degeneration; dementia of the Alzheimer type; disease/disorder; filamentous tau inclusion; gene product; interest; microtubule associated protein tau; microtubule associated protein tau aggregation; microtubule associated protein tau deposit; microtubule bound tau; microtubule-associated protein tau; microtubule-associated protein tau aggregation; microtubule-associated protein tau deposit; microtubule-bound tau; neurodegenerative illness; neurofibrillary degeneration; neurofibrillary lesion; neurofibrillary pathology; neuronal; paired helical filament of tau; primary degenerative dementia; self assembly; self-aggregate tau; senile dementia of the Alzheimer type; tangle; tau; tau PHF; tau PHF formation; tau Proteins; tau accumulation; tau aggregate; tau aggregation; tau associated neurodegeneration; tau associated neurodegenerative process; tau factor; tau fibrillization; tau filament; tau filament assembly; tau induced neurodegeneration; tau mediated neurodegeneration; tau neurodegenerative disease; tau neurofibrillary tangle; tau neuropathology; tau oligomer; tau paired helical filament; tau paired helical filament formation; tau polymerization; tau self-aggregate; tau-tau interaction; tauopathic neurodegenerative disorder; tauopathy

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinesin-1 is a motor protein that hydrolyzes ATP to drive the transport of intracellular cargo. It is composed of two heavy chains (KHCs) and two light chains (KLCs). Proper regulation of kinesin-1 prevents mislocalization of the motor and allows for coordination with other molecular motors. Regulated kinesin-1 is folded in half so that the KLCs and regulatory KHC tail domains come in contact with the enzymatically active KHC heads. This KHC/KLC complex is central to the regulatory mechanism of kinesin-1, yet the structure remains undetermined because the complex is not amenable to traditional structure determination techniques. To overcome this, we will use small-angle X-ray scattering (SAXS) to visualize the never-before-seen structure of a regulated kinesin-1 holoenzyme in solution at ~3nm resolution. Active kinesin-1 has significant structural variability, and we expect that only a radius of gyration for the entire extended molecule will be obtained. However, the regulated conformation appears to be a compact and rigid structure, therefore, we expect to obtain valuable information about the molecular shape of regulated kinesin-1 from SAXS. Together with chemical crosslinking studies, we will use the SAXS structure to determine where the various kinesin-1 elements interact with each other in this regulated complex. During our first session, we were able to collect sample data but subsequent analysis determined that the camera length was too short for optimum structure determination. We are scheduled to repeat our experiments with a longer camera on Nov 21, and we anticipate that this will yield the desired data.

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.