In Vitro Characterization of F646S-STAT5b Mutant: A Bioinactive STAT5b Protein

PAULA A, SCAGLIA; ALICIA S. MARTÍNEZ; EVA FEIGERLOVA; PENG FANG; MARIA GABRIELA BALLERINI; HÉCTOR G. JASPER; JUAN J HEINRICH; RON G. ROSENFELD; VIVIAN HWA; HORACIO M. DOMENÉ

Cartagena de Indias

Congreso; XXII Annual Meeting of the Sociedad Latinoamericana de Endocrinología Pediátrica (SLEP); 2011

Sociedad Latinoamericana de Endocrinología Pediátrica (SLEP)

In Vitro Characterization of F646S-STAT5b Mutant: A Bioinactive STAT5b Protein Associated with Severe Post Receptor Growth Hormone Insensitivity (GHI) and Immune Dysfunction. P. Scaglia1, A. Martínez1, E. Feigerlova2, P. Fang2,M. Ballerini1, H. Jasper1, J. Heinrich1, R. Rosenfeld2,V. Hwa2, H. Domené1 ¹Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hospital de Niños R. Gutiérrez Centro de Investigaciones Endocrinológicas (CEDIE), Buenos Aires, Argentina; División de Endocrinología; ²Oregon Health and Science, University- Department of Pediatrics, USA Introduction: Patients with STAT5b deficiency show growth hormone insensitivity and a variable degree of immune dysregulation. Objective: To characterize in vitro expression and functional transcriptional activity of the F646S-STAT5b mutant, found in a patient with severe GHI and immune dysfunction. Methods: FLAG-Tagged wild type (WT) and mutant F646S STAT5b, were transiently transfected into HEK293(hGHR) cell line. Functional studies included immunoprecipitation, Western immunoblot and immune detection of phosphorylated STAT5b proteins upon GH treatment. Luciferase (pGHRE-LUC)-transfected HEK293(hGHR) cells were analyzed for reporter activity. Results: In transiently expressed HEK293(hGHR) cell lines, expression of the F646S- STAT5b mutant was not as robust as WT. Upon GH treatment, F646S mutant can still be tyrosine phosphorylated, although to a lesser extent than WT. Immunologically equivalent concentrations of mutant F646-STAT5b protein showed no luciferase activity in contrast to a robust 100 fold induction of WT-STAT5b protein. Conclusions: In vitro characterization demonstrates that the F646S-STAT5b mutant retains some ability to become phosphorylated but is completely devoid of transcriptional activity. These findings suggest that this mutant results in a bioinactive STAT5b protein, responsible for complete GHI.