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 detailed molecular mechanisms of Stat activation, particularly the role of post-translational modifications and co-operation with cellular transcription factors are subject to intense investigation. The phosphorylation of a tyrosine residue in the carboxyl terminal domain is a common characteristic for the biologically active state of all known Stats. We studied the biological potential of purified recombinant murine Stat5a and Stat5b. These proteins were expressed in Sf9 insect cells upon infection with Stat5 encoding baculoviruses. We also obtained the tyrosine phosphorylated, activated forms of 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 specifically in vitro to the Stat5 DNA response element. This activated form of Stat5 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 phosphatase or with potato acidic phosphatase, which removes phosphate groups from serine and tyrosine residues. Phosphatase treatment resulted in the loss 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 potential of Stat5. This modification is, therefore, a sufficient prerequisite for transcriptional induction by Stat5.
Beisenherz-Huss
C
C
Institute for Anatomy, University of Freiburg, Albertstr. 23, D-79104 Freiburg, Germany.
Mundt
M
M
Herrala
A
A
Vihko
P
P
Schubert
A
A
Groner
B
B
D000818:Animals; D019556:COS Cells; D002845:Chromatography; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D008894:Milk Proteins; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008894:Milk Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D004247:DNA; D066246:ErbB Receptors; C099852:PDGF receptor tyrosine kinase; D017479:Receptors, Platelet-Derived Growth Factor; D017027:Protein Tyrosine Phosphatases