Localization of the TIG3 Transglutaminase Interaction Domain and Demonstration that the Amino-Terminal Region Is Required for TIG3 Function as a Keratinocyte Differentiation Regulator

Abstract

Tazarotene-induced gene 3 (TIG3) regulates keratinocyte terminal differentiation by activating type I transglutaminase (TG1). TIG3 consists of an amino-terminal (N-terminal) segment, that encodes several conserved motifs, and a carboxy-terminal (C-terminal) membrane-anchoring domain. By producing a series of truncation mutants that remove segments of the N-terminal region, and monitoring the ability of each mutant to co-precipitate TG1, function as a TG1 substrate, or functionally localize with TG1 in cells, we show that the TIG3 domain that interacts with TG1 is located within a TIG3 segment spanning amino acids 112-164. Although they bind TG1, TIG3 mutants lacking the conserved N-terminal region drive apoptosis-like cell death characterized by cell rounding, membrane blebbing, cytochrome c release, procaspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage, and reduced p53 and p21 levels. Compared with TIG3, these truncated mutants have an increased tendency to associate with membranes. A mutant lacking the C-terminal membrane-anchoring domain is inactive. These findings suggest that TIG3 interaction with TG1 does not require the N-terminal conserved domains, that the TIG3 N-terminal region is required for TIG3-dependent keratinocyte differentiation, that its removal converts TIG3 into a proapoptotic protein, and that this change in action of TIG3 is associated with an intracellular redistribution.

TIG3 Interaction at the Centrosome Alters Microtubule Distribution and Centrosome Function

“…Mutagenesis studies indicate that TIG3 mutants lacking the C-terminal membrane-anchoring domain are not active (Deucher et al, 2000;Sturniolo et al, 2003;Sturniolo et al, 2005). By contrast, N-terminal truncation converts TIG3 into a protein that causes keratinocyte apoptosis (Jans et al, 2008). TIG3 also suppresses the proliferation of keratinocytes (Sturniolo et al, 2003;Sturniolo et al, 2005), although very little is known about the mechanism of suppression.…”

“…1A shows that a mutant lacking the C-terminus, TIG3 (1-134), displays a diffuse cytoplasmic distribution and does not display perinuclear accumulation. We have not studied this mutant further in the present studies, because it is inactive and has no ability to modulate cell function (Jans et al, 2008;Sturniolo et al, 2003;Sturniolo et al, 2005). Because TIG3 suppresses cell proliferation, we are particularly interested in the intense staining that is observed adjacent to the nucleus (Fig.…”

“…TIG3 is present at high levels in differentiated human keratinocytes in suprabasal epidermis and in raft cultures (Jans et al, 2008). TIG3-dependent death is associated with activation of selected differentiation-associated processes.…”

RARRES3 (retinoic acid receptor responder (tazarotene induced) 3)

“…Localization of TIG3 to the centrosome is believed to be responsible for this decrease in cell survival, which leads to an increase in p21 level, a G1/S phase block, an activation of the caspase cascade, and a reorganization of the microtubule network (Scharadin et al, 2011). In contrast, expression of TIG3 in normal keratinocytes induces a process of terminal differentiation through the binding to and activation of type I transglutaminase and increase in cornified envelope formation to decrease cell survival (Sturniolo et al, 2003;Sturniolo et al, 2005;Jans et al, 2008). Localization of TIG3 to the cell membrane in keratinocytes is necessary for it to bind type I transglutaminase.…”

“…Removal of this domain from TIG3 causes it to distribute diffusely throughout the cytoplasm and reduces its ability to decrease cell survival Sturniolo et al, 2003;Sturniolo et al, 2005;Jans et al, 2008;Tsai et al, 2009 …”