Centrosomes play a critical role in establishing bipolar spindles. For the fidelity of cell division centrosomes must duplicate precisely once per cell cycle. Errors in this process result in mis- segregation of chromosomes. Aberrant centrosomes are often associated with genomic instability, a feature of many cancers. The proposed research uses the C. elegans embryo as an in vivo model to perform genetics-phosphoproteomic analyses of centrosome assembly. Among 5 essential centrosome factors in C. elegans, SAS-5 plays a key role in the assembly of new centrosomes, and its functional homologs (Ana2 and SIL/STIL) are also required for mitotic spindle organization. A mutation in the putative human homolog (SIL/STIL) of SAS-5 is linked to primary microcephaly (MCPH), an autosomal- recessive congenital disorder with reduced brain size. While we realize the great impact of SAS-5 for cell division and brain development, the molecular and biochemical mechanisms by which SAS-5 regulates centrosome assembly remain elusive. Our long-term goal is to elucidate the molecular and genetic mechanisms of the centrosome assembly. The objective is to understand regulatory mechanism of SAS-5 in centrosome assembly. Proper levels of centrosome proteins (SAS-6, and Plk4/Sak) are critical for the correct number of centrosomes, which is regulated by proteasomal destruction. Recent work proposed that protein phosphatase 2A (PP2A) targets SAS-5 to regulate centrosome assembly. Our central hypothesis is that SAS-5 is regulated by PP2A-dependent phosphorylation, which directs SAS-5 to 26S proteasome to ensure its proper level and localization. Our rationale is that identifying the sites dephosphorylated by PP2A and defining their critical role will reveal how site-specific phosphorylation events contribute to the regulation of SAS-5 activity and the fidelity of the centrosome assembly. We plan to test our central hypothesis by pursuing the following two specific aims: 1) Identify all phosphorylation sites of SAS-5 and specify the sites that are targeted by protein phosphatase 2A (PP2A). 2) Determine the biological impact of site-specific phosphorylation on SAS-5 in centrosome assembly. Toward these aims, we will use genetics, biochemistry, high- resolution imaging, and phosphoproteomics. We expect to identify phosphorylation sites of SAS-5 and their physiological roles responsible for proper activity of SAS-5 in centrosome assembly. The proposed research is significant, because understanding the mechanisms of centrosome assembly will likely provide insight relevant to the diagnosis and treatment of human diseases such as cancers and ciliopathies that are associated with centrioles/basal bodies, in addition to fundamentally advancing the field of centrosome biology.
The proposed research is relevant to public health because the molecular and biochemical mechanisms of regulated levels of centrosome proteins in C. elegans are expected to enhance our knowledge of centrosome regulation in human diseases with broad and important healthcare ramifications related to cancers and ciliopathies. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing knowledge that will help developing diagnostic and therapeutic inventions for human diseases.