Perk Protein Kinase Facilities Skin Regeneration via Association with Adhesion Molecules Independent of Its Catalytic Activity

Abstract

The epidermis protects the body from environmental and mechanical insults. When the skin is injured, healing requires re-epithelialization by keratinocytes through a coordinated migratory process known as keratinocyte collective cell migration (KCCM). Diseases such as diabetes often fail to progress to this stage of cutaneous wound healing, resulting in chronic non-healing wounds. Therefore, it is critical to understand the biochemical and molecular mechanisms of KCCM to develop novel therapeutics for treating chronic wounds. Cutaneous wounding triggers the Integrated Stress Response (ISR). The ISR is a cellular pathway that monitors cellular stresses by multiple sensory protein kinases, including PERK (EIF2AK3) and GCN2 (EIF2K4), which phosphorylate the eIF2, thereby mitigating translational control to alleviate stress-induced damage. We previously described that GCN2 facilitates KCCM via sustained phosphorylation of eIF2 and coordinated production of reactive oxygen species and amino acid transport. I have now discovered that PERK plays a crucial role in KCCM via mechanisms that are distinct from GCN2 and the ISR. Deletion of PERK via CRISPR methods resulted in altered keratinocyte morphology, impaired skin differentiation and stratification, and impaired KCCM, indicating the importance of PERK in skin homeostasis. Pharmacological and genetic rescue experiments showed that PERK protein kinase function is dispensable for KCCM, but rather PERK promotes the collective migration by scaffolding or tethering processes requiring the PERK cytoplasmic domain and its association to the ER. To delineate proteins that associate with PERK, I pursued BioID interactome analyses using an UltraID-tagged PERK and identified cytoskeletal and cell adhesion proteins as critical PERK targets. Based on these results, I observed that PERK-KO cells exhibit disrupted F-actin organization and impaired lamellipodia formation at the leading edge during KCCM. PERK-deleted cells showed impaired expression and localization of cell adhesion proteins, concomitant with elevated cell-substrate and cell-cell adhesions. Confocal microscopy and co-immunoprecipitation studies revealed that PERK interacts with the expression of the hemidesmosome proteins ITGA6, ITGB4, and COLXVII, as well as the desmosome proteins JUP, DSG2, and DSG3. These results indicate that PERK participates in multiple scaffolding functions with cell adhesion complexes that are crucial for establishing and maintaining skin homeostasis and keratinocyte migration.