Janus Kinase 3 Regulates Interleukin 2-induced Mucosal Wound Repair through Tyrosine Phosphorylation of Villin

Abstract

Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cellspecific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin.

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

“…2A, Jak3-V484* was tyrosine-phosphorylated both in vitro using the TKX1 expression system (first and second panels) and in stably transfected mammalian cells (HT-29Cl-19A) upon IL-2 activation (third and fourth panels). Previously, we showed that IL-2 treatment led to tyrosine phosphorylation of Jak3 in HT-29cl19A cells that facilitated its interactions with villin that enhanced IL-2-induced wound repair (8). Here we showed that IL-2 treatment also led to tyrosine phosphorylation of Jak3-V484* that co-immunoprecipitated with villin only in the presence of IL-2 (Fig.…”

“…1A) was done using pY20 (the top panel) and anti-GST (second from the top) antibodies. Tyrosine phosphorylation of Jak3-V484* in human IEC was determined by stable transfection of pCDNA-HA-Jak3-V484* into HT-29 Cl-19A cells treated with or without 50 units/ml of IL-2 for 15 min as reported before (8). Equal amounts of proteins from the cell lysates were subjected to IP using anti-HA antibody followed by IB using either pY20 (third from the top) or anti-HA (fourth from the top) antibody.…”

“…Methods for culture maintenance, IL-2 treatment, wound closure, and cell proliferations were reported before (8,9).…”

“…Villin knock-out mice showed compromised mucosal wound repair (11), and Jak3 mediated tyrosine phosphorylation of villin was necessary for its role in wound repair (8). However, the molecular mechanism of Jak3 interactions with villin is not known.…”

“…The crystal structure of the JH1 kinase domain has been solved (PDB ID:1 YVJ) but there is no report on the functions and/or crystal structure of other domains of Jak3. Previously, we reported that intestinal epithelial cells (IECs) express functional specific Jak3 (8,9), whose biological functions had been presumed to be largely limited to lymphocyte and macrophage populations (1), and proposed mechanisms through which activated Jak3 regulated mucosal wound repair through its interactions with cytoskeletal protein villin (8) and mucosal homeostasis (9).…”