The ubiquitin ligase HERC1 regulates cell migration via RAF-dependent regulation of MKK3/p38 signaling

Abstract

Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration. Post-translational modifications can modify proteins and regulate their functions. Protein ubiquitylation is a post-translational modification that covalently attaches ubiquitin to target proteins, altering protein function in an extraordinary variety of ways. This process occurs in three sequential steps: ubiquitin activation by a ubiquitin-activating enzyme (E1); transfer of activated ubiquitin from E1 to ubiquitin-conjugating enzyme (E2); and conjugation of ubiquitin to a lysine residue of a substrate protein by ubiquitin ligase enzyme (E3). This catalytic process can be repeated over several cycles, resulting in substrates modified on multiple lysine residues with different poly-ubiquitin chains. Since E3 ligases control substrate specificity and the ubiquitylation topology, they are emerging as key regulators of cellular processes 1-3. Cell migration plays a fundamental role in multiple physiological and pathological processes, including wound healing, embryogenesis, tissue morphogenesis, cancer metastasis, and inflammation. Numerous studies have demonstrated that mitogen-activated protein kinase (MAPK) pathways, including the Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK) signaling pathways, regulate cell migration via different mechanisms. Thus, the JNK signaling pathway modulates cell migration by phosphorylating proteins such as paxillin, DCX, Jun, and microtubule-associated proteins. Meanwhile, the p38 signaling pathway regulates migration by phosphorylating MAPK-activated protein kinase 2 (MAPKAP-K2 or MK2) and the ERK signaling pathway controls cell movement by phosphorylating myosin light chain kinase, calpain, or focal-adhesion kinase 4,5. Several ubiquitin E3 ligases also regulate cell migration by ubiquitylating the key proteins that control stress fiber form...

HERC1 Regulates Breast Cancer Cells Migration and Invasion

“…Our in vitro validation experiments showed that HERC1 depletion did not affect cell proliferation but directly inhibited cellular migration in Boyden chamber and wound healing assays, as well as invasion. In the course of our studies, Herc1 was reported as a regulator of migration, further supporting our findings [44].…”

HERC1 regulates breast cancer cells migration and invasion

“…Our in vitro validation experiments showed that HERC1 depletion did not affect cell proliferation but directly inhibited cellular migration in Boyden chamber and wound healing assays, as well as invasion. In the course of our studies, Herc1 was reported as a regulator of migration, further supporting our findings (34).…”

Serum amyloid A inhibits astrocyte migration via activating p38 MAPK

“…However, studies have also found that activating p38 promotes cell migration. In the ubiquitin ligase HERC1-depleted cells, for example, activation of p38 increases the migration of human osteosarcoma U2OS cells [52]. These findings suggest that various stimulants affect cell migration through specific signaling pathways.…”