IN-VITRO INTERACTION BETWEEN STAT5 AND JAK2 - DEPENDENCE UPON PHOSPHORYLATION STATUS OF STAT5 AND JAK2

A working model for haematopoietic cytokine signal transduction has be en hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associat ed members of the Janus kinase family. This is followed by tyrosine ph osphorylation of the associated receptor and members of the STAT (sign al transducer and activator of transcription) family of DNA-binding tr anscription factors. Phosphorylation is accompanied by STAT dimerisati on, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to pa lindromic STAT response elements. A number of areas of confusion remai n; not least the mechanism by which multiple cytokines signal via a li mited number of STATs. A role has been suggested for phosphorylated re ceptor tyrosine residues as STAT docking sites on activated receptor-J AK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activa tion. There is some controversy as to whether this model applies to ST AT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the c orrect molecular weight was expressed and found to be phosphorylated o n tyrosine residues and to bind to a STAT response DNA element. This b inding was dependent upon the phosphorylation status of the STAT prote in. DNA binding could be abolished in vitro by treatment with a phosph otyrosine phosphatase and restored in vitro by treatment with activate d recombinant JAK 2. The protein was purified to near homogeneity usin g a simple ion exchange/gel filtration chromatography procedure. The i nteraction between purified recombinant STAT 5 and JAK 2, either expre ssed by baculovirus or endogenously expressed in Buffalo rat liver cel ls, was studied. In both cases STAT 5 in its non-phosphorylated form w as found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the a ctivation of STAT 5 by a wide range of cytokines capable of activating JAK 2. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.