The candidate tumor suppressor gene, ING1, encodes several protein isoforms as a result of alternative splicing that may possess agonistic and antagonistic roles in the control of cell proliferation and apoptosis. Recently a related gene, ING2, was isolated in human whose expression is increased in adenocarcinomas. Little is known about the cellular function and regulation of these ING family members, but the fact that ING proteins contain a plant homeodomain finger suggests that these proteins may modulate transcription factor-mediated pathways. To elucidate how ING may interact in different tissues to modulate function, we used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates tissue-specific proliferation, differentiation, and apoptosis. We have isolated the first Xenopus laevis ING2 and demonstrate that transcript levels increase in response to TH treatment. We provide evidence for the existence of splice variants that are differentially expressed in tissues with different TH-induced fates. Western blots using an antibody directed against the highly conserved C-terminal end of ING proteins reveal a tissue-specific pattern of ING isoform expression in adult Xenopus tissues. Analyses of premetamorphic tadpole tissues show a TH-induced accumulation of ING proteins in tail, whereas the levels in the leg are not affected. This TH-induced accumulation is also observed in serum-free tail organ cultures and is prevented by inhibitors of tail apoptosis. Therefore, this work presents the first link between ING expression and a hormonally regulated nuclear transcription factor-mediated apoptotic response opening the possibility that ING family members may be involved in transducing the signal initiated by TH that determines cell fate. 2 (The former gene has been referred to as INGL and ING2, whereas the latter has been referred to as ING2 but is distinct from the former.) We will refer to the former gene as ING2 and the latter as ING4 to avoid confusion. A more distantly related ING3 mRNA encoding a putative 47-kDa protein has also been reported in mouse (GenBank TM accession number AY007790).3 ING2 maps to a different chromosome than ING1. Its putative protein product is predicted to also be 33 kDa in size and has 54% sequence identity to p33 ING1b (19,21 ING proteins belong to a family of plant homeodomain (PHD)

No abstract

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic ␣-chain and an associated ␤-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the ␣-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GII␣ is capable of associating with CD45 in a competitive situation where multiple GII␣ isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GII␣ acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.CD45 is a highly abundant, transmembrane, protein-tyrosine phosphatase expressed on all cells of hematopoietic origin (1). The cytoplasmic phosphatase activity of CD45 has been shown to be essential for the early signal transduction events leading to both thymocyte maturation and T cell activation (1). There is substantial evidence to suggest that CD45 regulates the tyrosine phosphorylation of Src family kinases (2, 3). The external domain of CD45 is extremely heterogeneous with respect to size and carbohydrate content primarily because of three alternatively spliced exons that encode for potential Olinked glycosylations (4). The usage of these exons appears to be developmentally regulated (4). As well, the extracellular domain encodes attachment sites for numerous N-linked glycans, and these glycosylations appear to be important for cell surface expression and protein stability of CD45 (5). Finally, although CD45 is a cell surface protein, no specific ligand for the extracellular domain has been definitively identified. Perhaps relevant to the present study, there have been studies suggesting that some lectins such as CD22 (6, 7), galectin-1 (8, 9), and the mannan-binding protein (10) are able to bind to CD45 carbohydrate, although the biological significance of these interactions are largely not understood.Recently, our laboratory has demonstrated that the carbohydrate processing enzyme ␣-glucosidase II (GII) 1 physically interacts with CD45 (11). GII is found within the ER and catalyzes the hydrolysis of the inner two ␣1,3-linked glucose residues present on all N-linked immature oligosaccharides (12, 13). This processing of glucose in the ER has been shown to be intimately involved in protein folding by regulating the interaction between the nascent polypeptide and the lectin chaperones calnexin and calreticulin. More specifically, removal of t...