Constitutive activation of STAT5 by a point mutation in the SH2 domain.
We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.
Ariyoshi
K
K
Department of Hematopoietic Factors, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Nosaka
T
T
Yamada
K
K
Onishi
M
M
Oka
Y
Y
Miyajima
A
A
Kitamura
T
T
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D019556:COS Cells; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D008894:Milk Proteins; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D018909:src Homology Domains
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D008894:Milk Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor