title: Functional analysis of the nuclear localization signal of the POU transcription factor Skn‑1a in epidermal keratinocytes.
abstract: POU domain proteins are a family of critical regulators of development and differentiation due to their transcriptional activity in the nucleus. Skn‑1a, a member of the POU domain protein family, appears to be expressed predominantly in epidermal keratinocytes and is thought to play a critical role in keratinocyte differentiation and proliferation. In this study, we examined the mechanisms involved in the nuclear localization of Skn‑1a. We transiently expressed enhanced green fluorescent protein (EGFP) reporter constructs encoding EGFP fusions with Skn‑1a deletion and mutation proteins in normal human epidermal keratinocytes (NHEKs). The experiments clearly demonstrated that Skn‑1a contained a functional nuclear localization signal (NLS) domain, and that the smallest domain necessary for Skn‑1a nuclear transport was the GRKRKKR sequence located within amino acids 279‑285. Previous studies have shown that the phosphorylation of specific amino acids neighboring the NLS may regulate nuclear transport and that the amino acid residues threonine (Thr) and serine (Ser) have the potential to undergo phosphorylation. We examined whether the amino acids Thr286 and Ser287, which reside adjacent to the NLS at the carboxy‑terminal side, play a role in Skn‑1a nuclear localization. For this purpose, we generated three EGFP‑Skn‑1a mutation constructs, in which Thr286, Ser287, or both Thr286 and Ser287 residues were replaced with alanine, respectively. The results showed that the Thr286 and Ser287 residues were involved in the regulation of nuclear localization as well as epidermal differentiation. These results suggested that the epidermal differentiation signaling pathway, involving kinase and phosphatase activation, may regulate the NLS activity of Skn‑1a in keratinocytes. Collectively, these data contribute to understanding the mechanisms of nuclear translocation of POU domain proteins and epidermal differentiation.
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Ryuta
authors: R
authors: Moritsugu
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Katsuto
authors: K
authors: Tamai
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Hajime
authors: H
authors: Nakano
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Takayuki
authors: T
authors: Aizu
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Koji
authors: K
authors: Nakajima
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Takehiro
authors: T
authors: Yamazaki
authors: Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562, Japan.
authors: Daisuke
authors: D
authors: Sawamura
mesh_terms: D021581:Active Transport, Cell Nucleus; D002454:Cell Differentiation / Q000235:genetics*; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics*; D050810:Octamer Transcription Factors / Q000235:genetics* / Q000378:metabolism; D050809:POU Domain Factors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics
chemical_list: D019913:Nuclear Localization Signals; D050810:Octamer Transcription Factors; D050809:POU Domain Factors; C484169:POU2F3 protein, human; D049452:Green Fluorescent Proteins