THE BLOCKADE OF PREADIPOCYTE DIFFERENTIATION BY PROTEIN-TYROSINE-PHOSPHATASE HA2 IS REVERSED BY VANADATE

A tyrosine phosphatase, i.e, PTPase HA2, was previously isolated from 3T3-L1 cells and characterized using O-phospho Tyrosine(19)-422/aP2 protein (a target of the insulin receptor tyrosine kinase) as substrate. The nucleotide sequence of a PTPase HA2 cDNA showed it to be a homologue of PTPase 1B. When induced to differentiate into adipocytes, confluent 3T3-L1 preadipocytes undergo mitotic clonal expansion followed by growth arrest and then coordinate expression of adipocyte genes, During clonal expansion, expression of PTPase HA2 increases abruptly and then decreases concomitant with the transcriptional activation of adipocyte genes, Constitutive expression of the PTPase by 3T3-L1 preadipocytes using a PTPase HA2 expression vector prevents adipocyte gene expression and differentiation into adipocytes, Appropriately timed exposure of transfected preadipocytes to vanadate (a PTPase inhibitor), just as clonal expansion ceases restores their capacity to differentiate, Treatment of transfected preadipocytes with vanadate prior to or during clonal expansion fails to reverse PTPase HA2-blocked differentiation, whereas treatment of untransfected preadipocytes during mitotic clonal expansion blocks differentiation. Vanadate added following clonal expansion has no effect on differentiation. Thus, a critical tyrosine phosphorylation event(s) occurs between termination of clonal expansion and initiation of adipocyte gene expression while a critical tyrosine dephosphorylation event(s) occurs during clonal expansion.