Dataset Information

LC-MS/MS analysis of FLAG-NEK9 immunoprecipitates

ABSTRACT: Immunoprecipitates of FLAG-NEK9 was analyzed to detect interacting proteins under serum starvation conditions.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Early Embryonic Cell

DISEASE(S): Disease Free

SUBMITTER: Yasuhiro Yamamoto

LAB HEAD: Noboru Mizushima

PROVIDER: PXD024292 | Pride | 2021-04-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	LC-MSMS_of_FLAG-NEK9_raw.xlsx	Xlsx
	Table_S2.xlsx	Xlsx
	checksum.txt	Txt

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Publications

NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9/myosin IIA.

Yamamoto Yasuhiro Y Chino Haruka H Tsukamoto Satoshi S Ode Koji L KL Ueda Hiroki R HR Mizushima Noboru N

Nature communications 20210602 1

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis t ...[more]

PMID: 34078910

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Project description:Cilia assembly, maintenance and the localization of signal-transduction proteins necessitate a functioning intraflagellar transport (IFT). The IFT machinery moves cargo along a microtubule scaffold from the proximal to the distal end of the cilium. This anterograde transport is facilitated by a large multiprotein complex IFT-B, which consists of 16 proteins in total and recruits motor protein Kinesin-2 for active movement. TTC30A and TTC30B are integral components of this complex and were shown before to have redundant function in context of IFT. One paralogue could compensate the loss of the other, preventing the disruption of IFT-B and thus a severe ciliogenesis defect with no formation of the cilium. Affinity-based protein complex analysis of endogenously tagged cells, generated via CRISPR/Cas9 system as described before, revealed paralogue specific protein interactors proposing the involvement of TTC30A or TTC30B in other ciliary functions, particularly Sonic hedgehog signaling (Shh). Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which intriguingly is also linked to a rare TTC30 variant. In this study we focus on the so far unknown interaction of TTC30A with protein kinase A catalytic subunit α PRKACA, which is a negative regulator of Shh pathway. For an in-depth analysis of this unique interaction and the influence on Shh, we used previously, via the CRISPR/Cas9 system, generated TTC30A or B single- and double-knockout hTERT-RPE1 cells as well as rescue cells harboring the TTC30 mutation. Our systematic approach revealed the paralogue specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA as well as the uptake of Smoothened into the cilium resulting in a downregulation of Sonic hedgehog signaling. Affinity-based protein complex analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in interaction pattern. These interactome alterations combined with Shh downregulation suggest a possible mechanism of how mutant TTC30A and respectively TTC30A KO are linked to synpolydactyly.