RIPK4 activity in keratinocytes is controlled by the SCFβ-TrCP ubiquitin ligase to maintain cortical actin organization

Abstract

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-κB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCF ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCF complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of β-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. β-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.

Insight Into the Function of RIPK4 in Keratinocyte Differentiation and Carcinogenesis

“…PKCs could activate RIPK4, which then induced uncontrolled epidermal inflammatory responses (17). Transgenic mice with epidermal overexpressing RIPK4 reacted hypersensitively to PKC (12,17). This was in accordance with the finding that RIPK4 knockdown in normal keratinocytes hampered the expression of differentiation markers upon PKC activation (18).…”

“…However, understanding the activation mechanism of RIPK4 gives valuable information about how RIPK4 functions at the biochemical level and how it controls epidermal differentiation and carcinogenesis. Constitutively activated RIPK4 by PKC can be degraded by the SCF β– T r C P -mediated proteasomal degradation pathway to maintain cortical actin organization in cultured keratinocytes ( 17 ). β-TrCP, which regulates multiple signaling pathways controlling cell growth, survival, death, and differentiation, is found to be involved in the process of carcinogenesis ( 66 , 67 ).…”

“…As previously mentioned, RIPK4 was initially identified as an interaction partner of the PKC family. PKCs could activate RIPK4, which then induced uncontrolled epidermal inflammatory responses (17). Transgenic mice with epidermal overexpressing RIPK4 reacted hypersensitively to PKC (12,17).…”

RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

“…However, in contrast to HaCaT cells, si‐RNA mediated depletion of endogenous RIPK4 in the HepG2 cell line did not show any effects on TGF‐β1‐induced 3TP‐Lux and ARE‐Lux reporter gene expression (Figure B, iii‐iv). It has been demonstrated that in non‐keratinocyte cell lines, RIPK4 was in the non‐phosphorylated (non‐active) form, in contrast to keratinocyte cell lines in which RIPK4 was present in both auto‐phosphorylated (active) and non‐phosphorylated (non‐active) forms (Tanghe et al, ). To evaluate the ineffectiveness of RIPK4 depletion on TGF‐β1‐induced 3TP‐Lux and ARE‐Lux reporter gene expression, we examined the phosphorylation status of overexpressed and endogenous RIPK4 protein in HepG2 cells along with HaCaT cells.…”

Defining the relevant combinatorial space of the PKC/CARD-CC signal transduction nodes

“…We would like to thank Dr. Corinne Rösselet and Prof. Dr. Wim Declercq, Ghent University, for gateway entry vectors with activated mutant PKCs [111]. pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138) [112].…”