A Mouse Homologue of the Drosophila Tumor Suppressor l(2)tid Gene Defines a Novel Ras GTPase-activating Protein (RasGAP)-binding Protein

Abstract

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1 L and hTid-1 S forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.

TID1, a mammalian homologue of the drosophila tumor suppressor lethal(2) tumorous imaginal discs, regulates activation-induced cell death in Th2 cells

“…This antibody specifically detects two proteins of 43 and 40 kDa that comigrate on SDS-PAGE with human TidL and human TidS, respectively (Figure 1b). Hence, these two forms correspond to the major splice variants that have been published (Trentin et al, 2001). Consistent with this report there was no evidence for the expression of a third splice variant (Trentin et al, 2001;Yin and Rozakis-Adcock, 2001), supporting the notion that this form represents a low abundance mRNA species, and that the encoded protein may be highly unstable (Trentin et al, 2001).…”

“…The murine homologues of these splice variants, in particular their respective carboxyl termini, are highly conserved (Trentin et al, 2001) ( Figure 1a). Our previous experiments with TidL and TidS J-domain mutants suggested that these splice variants have distinct cellular targets.…”

“…Hence, these two forms correspond to the major splice variants that have been published (Trentin et al, 2001). Consistent with this report there was no evidence for the expression of a third splice variant (Trentin et al, 2001;Yin and Rozakis-Adcock, 2001), supporting the notion that this form represents a low abundance mRNA species, and that the encoded protein may be highly unstable (Trentin et al, 2001). Analysis of increasing amounts of NIH 3T3 cell extracts by SDS-PAGE, followed by immunoblotting and quantitation using a digital imaging system, showed that the ratio of the signal for murine TidL and murine TidS remained constant (Figure 1c), demonstrating that the human Tid-specific RS-13 monoclonal antibody (Syken et al, 1999) recognized the murine TidL and TidS splice variants with similar efficiencies.…”

Identification of a hTid‐1 mutation which sensitizes gliomas to apoptosis

“…Protein concentrations were determined using the Bio-Rad protein assay. A total of 50 lg of protein from each sample was then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane and subjected to Western blot analysis with rabbit anti-mouse Tid-1 antibody [5], mouse monoclonal anti-human PARP monoclonal antibody (C2-10; BD PharMingen), or monoclonal anti-Bcl-X L (H-62; Santa Cruz). Proteins were detected using ECL reagent (Amersham Pharmacia Biotech) according to the manufacturerÕs protocol.…”

“…Three alternatively spliced variants of the human TID1 gene have been described [3], the hTid-1 L and hTid-1 S being the most abundant [3][4][5]. Expression of the third and shortest splice variant is restricted to the thymus [3], and the corresponding endogenous 38-kDa protein has only been detected in cell lysates from human Thp1 and U937 leukemia lines [6].…”