Dataset Information

Fission yeast NDR/LATS kinase Orb6 regulates exocytosis via phosphorylation of exocyst complex

ABSTRACT: NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals, but few of their substrates are known. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity via spatial regulation of Gef1, a guanine nucleotide exchange factor for the small GTPase Cdc42. Here we show that Orb6 plays a critical role as a positive regulator of exocytosis, independent of Gef1. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify several proteins involved in membrane trafficking as Orb6 targets, and we confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro. Our results suggest that Orb6 kinase activity is crucial for exocyst localization to actively-growing cell tips and for exocyst activity during septum dissolution after cytokinesis. We further show that Orb6 phosphorylation of Sec3 serine-201 contributes to exocyst function in parallel with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Schizosaccharomyces Pombe 927

TISSUE(S): Cell Line Cell

SUBMITTER: Kenneth Sawin

LAB HEAD: Ken Sawin

PROVIDER: PXD009408 | Pride | 2019-01-31

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 115

Publications

Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex.

Tay Ye Dee YD Leda Marcin M Spanos Christos C Rappsilber Juri J Goryachev Andrew B AB Sawin Kenneth E KE

Cell reports 20190201 6

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in memb ...[more]

PMID: 30726745

Similar Datasets

Project description:In budding yeast, the Mitotic Exit Network (MEN), a GTPase signaling cascade integrates spatial and temporal signals to promote exit from mitosis. This signal integration requires transmission of a signal created on the cytoplasmic face of the spindle pole bodies (SPBs, functional equivalent of the centrosome in yeast) to the nucleolus, where the MEN effector protein Cdc14 resides. In this study, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation by the Polo kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Cdc5 phosphorylates the Cdc14 nucleolar anchor Cfi1/Net1 creating a phospho-binding site for Dbf2-Mob1 which allows Dbf2-Mob1 to phosphorylate Cfi1/Net1 and Cdc14, which activates Cdc14. The mechanisms governing intracellular signal transmission that we uncovered in the MEN – regulated nuclear access and effector activation through priming kinases - may well serve as paradigms for intracellular signal transmission in general. As for phosphoproteomics, our analyses suggest a simple model where Cdc5 and Dbf2-Mob1 phosphorylate Cfi1/Net1 to bring about the release of Cdc14 from its inhibitor. Previous studies have shown that Cfi1/Net1 is a highly phosphorylated protein. To map sites in Cfi1/Net1 that are phosphorylated in a CDC5 or MEN-dependent manner, we performed phosphoproteomics analyses on wild-type anaphase cells and cells in which Cdc5 or Cdc15 were inhibited. We synchronized wild-type, cdc5-as1 and cdc15-as1 cells in G1 with α-factor pheromone and released them into the cell cycle with the corresponding inhibitors. Cultures enriched in anaphase cells as judged by anaphase spindle formation (~70% for wild-type cells and ~95% for cdc5-as1 and cdc15-as1 cells) were collected and processed for a reproducible data-independent acquisition mass spectrometry (DIA-MS) analysis with 7 technical replicates for each sample. Also, 10 out of 12 CDC5-only sites identified in anaphase cells are already phosphorylated in cells arrested in metaphase using the microtubule depolymerizing drug nocodazole with 6 of them were also determined to be CDC5-dependent in metaphase, which is consistent with the finding that Cdc5 is already active in metaphase.

			Action	DRS
	GST-Sec3_IP_Rep1.raw	Raw
	GST-Sec3_IP_Rep1.zip	Other
	GST-Sec3_IP_Rep2.raw	Raw
	GST-Sec3_IP_Rep2.zip	Other
	GST-Sec3_IP_Rep3.raw	Raw