| Summary: | Cell signaling controls tissue homeostasis by balancing cell proliferation, differentiation, and cell death. In skin, tissue homeostasis is sustained by epidermal progenitor cells, which are localized within the basal layer of the skin epithelium. Posttranslational modifications of the cellular proteome, such as protein phosphorylation, play a fundamental role in the regulation of cell stemness and differentiation. However, it remains unclear how proteomic changes, specifically phosphorylation, occur and contribute to epidermal differentiation. Here, I present new findings that skin differentiation of basal skin keratinocytes is partially dependent on the kinase Receptor-interacting protein kinase 4 (RIPK4) and the desmosome protein Plakophilin-1 (Pkp-1). My results suggest RIPK4 phosphorylates Pkp-1, allowing phosphorylated Pkp-1 to bind to a scaffolding protein called Leucine-Rich Repeat Scaffold Protein (Shoc2) to disrupt MAPK kinase signaling and ultimately allow cell differentiation.
In this study, I used a quantitative phosphoproteomics technique called stable-isotope labeling of amino acids in cell culture (SILAC) to investigate which proteins are phosphorylated during skin differentiation. (Tables of the SILAC results can be found online as supplementary files. Supplementary Table 1 lists the phosphosites and SILAC ratios of undifferentiated keratinocytes compared to 12 hour differentiated keratinocytes. Supplementary Table 2 lists the phosphosites and SILAC ratios of differentiated wild-type keratinocytes compared to undifferentiated RIPK4 KO keratinocytes.) My results show desmosome proteins are highly phosphorylated during skin differentiation. To test whether Pkp-1 plays a role in this process, I used genome editing techniques and found that absence of Pkp-1 leads to impaired skin differentiation. Furthermore, loss of three phosphosites on Pkp-1, identified by SILAC, also caused impaired skin differentiation. This data suggests Pkp-1 protein is necessary for skin differentiation, specifically phosphorylated Pkp-1.
To identify the kinase that phosphorylates Pkp-1, we performed a mammalian kinome cDNA library screen and identified RIPK4 (receptor-interacting serine-threonine kinase 4) as a kinase of Pkp-1. Our in vitro kinase assay shows RIPK4 can phosphorylate Pkp-1. Additionally, my in vitro cell culture kinase assay results suggest RIPK4 can phosphorylate wild-type Pkp-1, while non-phosphorylatable Pkp-1 (Serine-to-alanine conversions at 3 phosphosites found in SILAC) cannot be phosphorylated by Pkp-1. This suggests RIPK4 is a key kinase regulating Pkp-1 phosphorylation during skin differentiation.
Previously, desmosome protein desmoglein1 was postulated to be required for in vitro skin keratinocyte differentiation. The research suggests Dsg1 interacts with Shoc2 and Erbin to facilitate skin differentiation. I postulated Pkp-1 may be a part of this signaling pathway. To test this, I performed immunoprecipitation of Pkp-1 and blotted for Dsg1, Shoc2, and Erbin. I find that Pkp-1 does not bind to Dsg1 nor Erbin, but does bind to Shoc2. Additionally, loss of Pkp-1 leads to increased phosphorylated ERK protein levels. Overall, my data suggests that phosphorylated Pkp-1, but not unphosphorylated Pkp-1, interacts with Shoc2 to decrease ERK phosphorylation and facilitate skin differentiation.
Taken together, through a comprehensive approach encompassing proteomics, mouse genetics, with other cell and molecular biology techniques, my research presents a global view of phosphoproteomic changes that occur during epidermal differentiation and identifies a novel signaling axis involved in skin differentiation.
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