Fission yeast CK1 promotes DNA double-strand break repair through both homologous recombination and non-homologous end joining.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1.

Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Here, we have investigated the localization and interactomes of endogenously tagged CK1δ and CK1ε during interphase and mitosis. CK1δ and CK1ε localize to centrosomes throughout the cell cycle, and in interphase cells to the nucleus, and in both a diffuse and punctate pattern in the cytoplasm. Also, for the first time, they were detected at the midbody during cell division. Mass spectrometry analysis identified a total of 181 proteins co-purifying with a Venus multifunctional (VM)-tagged CK1δ and/or CK1ε. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient endocytosis, was consistently one of the most abundant interacting partners. We demonstrate that GAPVD1 is a substrate of CK1δ/ε with up to 38 phosphorylated residues in vitro and in vivo. Wildtype and a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, were able to rescue defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1. Our results indicate that GAPVD1 is an important interacting partner and substrate of CK1δ/ε for endocytosis.

The kinase domain of CK1 enzymes contains the localization cue essential for compartmentalized signaling at the spindle pole.

CK1 protein kinases contribute to multiple biological processes, but how they are tailored to function in compartmentalized signaling events is largely unknown. Hhp1 and Hhp2 (Hhp1/2) are the soluble CK1 family members in Schizosaccharomyces pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. The SIN is assembled by Sid4 at spindle pole bodies (SPBs), and though Hhp1/2 colocalize there, it is not known how they are targeted there or whether their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize throughout the cell cycle to SPBs, as well as to the nucleus, cell tips, and division site. We find that their catalytic domains but not their enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the setting of spindle stress. Taken together, these data have enabled us to define a molecular mechanism used by CK1 enzymes to target a specific cellular locale for compartmentalized signaling.

An Asp-CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells.

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure-function analysis support this hypothesis. Disruption of the Asp-CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp's spindle function; however, it is completely dispensable for Asp's role in microcephaly suppression.

Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies.

Pericentrin is a critical centrosomal protein required for organizing pericentriolar material (PCM) in mitosis. Mutations in pericentrin cause the human genetic disorder Majewski/microcephalic osteodysplastic primordial dwarfism type II, making a detailed understanding of its regulation extremely important. Germaine to pericentrin's function in organizing PCM is its ability to localize to the centrosome through the conserved C-terminal PACT domain. Here we use Drosophila pericentrin-like-protein (PLP) to understand how the PACT domain is regulated. We show that the interaction of PLP with calmodulin (CaM) at two highly conserved CaM-binding sites in the PACT domain controls the proper targeting of PLP to the centrosome. Disrupting the PLP-CaM interaction with single point mutations renders PLP inefficient in localizing to centrioles in cultured S2 cells and Drosophila neuroblasts. Although levels of PCM are unaffected, it is highly disorganized. We also demonstrate that basal body formation in the male testes and the production of functional sperm does not rely on the PLP-CaM interaction, whereas production of functional mechanosensory neurons does.