A molecular mechanism for the procentriole recruitment of Ana2

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole "targeting-factor." In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phosphoresidues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4's kinase domain. Thus, Asl-A's phosphorylation state determines which of Asl-A's two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.National Cancer Institute [P30CA23074]; National Institute of General Medical Sciences [R01 GM110166, GM126035]; National Science Foundation [MCB-1158151]; Phoenix Friends of the University of Arizona Cancer CenterThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Vesicular trafficking plays a role in centriole disengagement and duplication

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/ Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.National Institute of General Medical Sciences (NIGMS) [R01GM074876, P30GM106397]; National Cancer Institute [P30 CA23074]; NIH/NIGMS [R01GFM110166, R01GM126035]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4

The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membranebound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a `domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes.Division of Intramural Research at the NIH/NHLBI [1ZIAHL006104]; NIH/NIGMS [R01GM110166, R01GM094415]; NSF [MCB1158151]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A molecular mechanism for the procentriole recruitment of Ana2

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 is then phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STil/ANa2 (STAN) domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N terminus of Ana2, which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.Division of Intramural Research at the National Heart, Lung, and Blood Institute [1ZIA HL006104]; National Cancer Institute [P30 CA23074]; National Institute of General Medical Sciences [R01GFM110166]; National Science Foundation [MCB1158151]; Phoenix Friends6 month embargo; published online: 1 March 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]