Specific DNA binding and transactivation potential of recombinant, purified Stat5

The signal transducers and activators of transcriptions (Stats) are central mediators of cytokine responses especially in hematopoietic cells. The det ailed molecular mechanisms of Stat activation, particularly the role of pos t-translational modifications and co-operation with cellular transcription factors are subject to intense investigation. The phosphorylation of a tyro sine residue in the carboxyl terminal domain is a common characteristic for the biologically active state of all known Stats. We studied the biologica l potential of purified recombinant murine Stat5a and Stat5b. These protein s were expressed in Sf9 insect cells upon infection with Stat5 encoding bac uloviruses. We also obtained the tyrosine phosphorylated, activated forms o f the Stat5 proteins by expressing the tyrosine kinase Janus kinase2 (Jak) in the same cells through co-infection with a kinase encoding virus. After purification, only the tyrosine phosphorylated form was able to bind specif ically in vitro to the Stat5 DNA response element. This activated form of S tat5 is also able to support specific cell free in vitro transcription of a gene with a Stat5 response element in its promoter region. The recombinant purified Stat5 proteins were treated with the tyrosine specific protein ph osphatase or with potato acidic phosphatase, which removes phosphate groups from serine and tyrosine residues. Phosphatase treatment resulted in the l oss of specific DNA binding ability. This property could be restored by an in vitro reaction with recombinant, purified EGF or PDGF receptor kinases. Tyrosine rephosphorylation in vitro also restored the transactivation poten tial of Stat5. This modification is, therefore. a sufficient prerequisite f or transcriptional induction by Stat5. (C) 2001 Elsevier Science Ireland Lt d. All rights reserved.