THE LAST PROLINE OF BOX-1 IS ESSENTIAL FOR ASSOCIATION WITH JAK2 AND FUNCTIONAL ACTIVATION OF THE PROLACTIN RECEPTOR

The interaction of prolactin (PRL) with its receptor leads to activati on of the tyrosine kinase, Janus kinase 2 (JAK2). In the cytoplasmic j uxtamembrane region, a short segment (Box 1) which is conserved in oth er receptors of the PRL/growth hormone (GH)/cytokine receptor family. is required for signal transduction. To assess the contribution of the different amino acids of Box 1, individual alanine substitutions of a ll residues, grouped substitution of four prolines (4PA mutant) and in dividual leucine replacement of the two last. prolines (P248L and P250 L mutants) were introduced. Here we show that P250L and 4PA (i) inhibi t PRL-induced transactivation of a luciferase reporter governed by a b eta -caseine gene promoter (ii) decrease in JAK2 tyrosine kinase activ ity in biotinylated-PRL precipitates; (iii) impair the interaction bet ween PRLR and JAK2, as evidenced by lack of co-immunoprecipitation: (i v) and prevent the activation of signal transducer and activator of tr anscription (Stat) as determined by absence of tyrosine phosphorylatio n of Stat5. Our data suggest that the Box 1 region of the PRL receptor and particularly the last proline is critical for JAK2 association an d subsequent activation. These results support the notion that the tyr osine kinase JAK2 is implicated in activation of downstream protein ef fectors such as Stat5, which are involved in transcription of PRL-resp onsive genes. (C) 1997 Elsevier Science Ireland Ltd.