Dataset Information

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation

ABSTRACT: Proteomic analysis revealed potential phospho-acceptor sites within Vpx by PIM3. HEK293 cells were transfected with plasmid vector encoding Vpx together with either empty vector or PIM3. Immunoprecipitated Vpx was subjected to liquid chromatography tandem-mass spectrometry.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akihide Ryo

PROVIDER: PXD013154 | JPOST Repository | Wed Mar 20 00:00:00 GMT 2019

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	150325_V175_3.mgf	Mgf
	150325_V175_3.raw	Raw
	F019474_0.csv	Csv

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Publications

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

Miyakawa Kei K Matsunaga Satoko S Yokoyama Masaru M Nomaguchi Masako M Kimura Yayoi Y Nishi Mayuko M Kimura Hirokazu H Sato Hironori H Hirano Hisashi H Tamura Tomohiko T Akari Hirofumi H Miura Tomoyuki T Adachi Akio A Sawasaki Tatsuya T Yamamoto Naoki N Ryo Akihide A

Nature communications 20190423 1

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PI ...[more]

PMID: 31015445

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Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.