Dataset Information

PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules independent of its kinase activity

ABSTRACT: Proximity labeling experiments (BioID) were performed on Human NTERT keratinocytes depleted of PERK (EIF2AK3, PERK-KO) re-expressing a WT PERK containing a C-terminal UltraID tag. PERK-KO and PERKUltraID cells were treated with differentiation medium containing 50 uM of exogenous Biotin for 6 h, washed three times with ice-cold PBS and harvested on ice with a modified RIPA buffer solution (25mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA, 1x HALT protease/phosphatase inhibitor cocktail). Samples were then sonicated and incubated at 4C with rotation for 30 minutes, followed by centrifugation step to pellet cell debris. Total protein concentration was measured, and the samples were normalized to 1 mg/ml to a final volume of 1 ml. Normalized samples were incubated with 40uL of trypsin-resistant streptavidin beads with rotation overnight at 4C. The next day, the beads were collected by magnetic separation and washed three times with an IP wash solution (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100), followed by two washes with PBS. Finally, the beads were resuspended with 100 ul of PBS and submitted on ice for preparation and mass spectrometry analysis by the Indiana University School of Medicine Center for Proteome Analysis.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ronald C. Wek Dan F. Spandau Amber L. Mosley

PROVIDER: MSV000098126 | MassIVE | Wed Jun 11 12:13:00 BST 2025

SECONDARY ACCESSION(S): PXD064891

REPOSITORIES: MassIVE

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PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules, independent of its kinase activity.

Diaz Miguel Barriera MB Staschke Kirk A KA Baucum Anthony J AJ Spandau Dan F DF Wek Ronald C RC

Molecular biology of the cell 20250924 11

Successful cutaneous wound healing requires reepithelialization by keratinocytes using a coordinated migratory process called keratinocyte collective cell migration (KCCM). Environmental stresses such as wounding induce the integrated stress response (ISR) initiated by protein kinases that phosphorylate the α subunit of eIF2 and mitigate translational control to alleviate stress damage. We previously reported that the ISR protein kinase GCN2 (EIF2AK4) facilitates KCCM via sustained phosphorylati ...[more]

PMID: 40991407

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