Abstract
Mitogenic signals that regulate cell division often proceed through multienzyme assemblies within defined intracellular compartments. The anchoring protein Gravin restricts the action of mitotic kinases and cell-cycle effectors to defined mitotic structures. In this report we discover that genetic deletion of Gravin disrupts proper accumulation and asymmetric distribution of γ-tubulin during mitosis. We utilize a new precision pharmacology tool, Local Kinase Inhibition, to inhibit the Gravin binding partner polo-like kinase 1 at spindle poles. Using a combination of gene-editing approaches, quantitative imaging, and biochemical assays, we provide evidence that disruption of local polo-like kinase 1 signaling underlies the γ-tubulin distribution defects observed with Gravin loss. Our study uncovers a new role for Gravin in coordinating γ-tubulin recruitment during mitosis and illuminates the mechanism by which signaling enzymes regulate this process at a distinct subcellular location.
Highlights
Spatial biology is an emerging aspect of biomedicine wherein investigators study how the subcellular location of enzymes underlies health and disease [1, 2]
Immunoblots of mouse embryonic fibroblasts (MEFs) from WT and Gravin null (2/2) mice established that total cellular protein expression of g-tubulin was similar for both genotypes (Fig. 1A)
Immunofluorescence detection confirmed that g-tubulin decorates spindle poles in WT MEFs during mitosis (Fig. 1B)
Summary
Spatial biology is an emerging aspect of biomedicine wherein investigators study how the subcellular location of enzymes underlies health and disease [1, 2]. Intracellular targeting of cell-signaling enzymes is achieved through the interaction with anchoring, adaptor, or scaffolding proteins [3]. Recent findings highlight that AKAPs provide spatial and temporal synchronization of protein kinases that control the mammalian cell cycle. Several multivalent AKAPs such as pericentrin, AKAP450, and Gravin have been implicated in targeting PKA and other signaling enzymes to mitotic structures [8,9,10,11]. During mitosis Gravin anchors Aurora A and polo-like kinase 1 (Plk1) at the spindle poles [12]. We discover that Gravin loss impairs the accumulation of g-tubulin at the spindle poles during mitosis. We utilize a recently developed precision pharmacology tool, Local Kinase Inhibition (LoKI), to demonstrate that targeted inhibition of centrosomal Plk alters the accumulation and asymmetric distribution of g-tubulin at mitotic spindle poles. We show that deletion of Gravin disrupts the formation of Nedd1/ g-tubulin sub-complexes that are necessary for tubulin ring assembly, illuminating a key centrosome-specific function of this anchoring protein
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