THE ROLE OF THE GROWTH-HORMONE (GH) RECEPTOR AND JAK1 AND JAK2 KINASES IN THE ACTIVATION OF STAT-1, STAT-3, AND STAT-5 BY GH

GH has been shown to activate the GH receptor (GHR)-associated tyrosin e kinase JAK2 and the Src homology 2 domain-containing transcription f actors Stats (signal transducers and activators of transcription) 1, 3 , and 5. The present work investigates the role of GHR and JAK2 in the activation of Stats 1, 3, and 5 by GH. The ability of GH to stimulate the tyrosyl phosphorylation of these Stats was assessed in Chinese ha mster ovary (CHO) cells expressing truncated and mutated GHR. GH was o bserved to stimulate tyrosyl phosphorylation of Stats 1, 3, and 5 in C HO cells expressing GHRs that bind JAK2 [GHR(1-638) (full-length) and GHR(1-454) (lacks approximately half of the cytoplasmic domain)] but n ot in CHO cells expressing GHR that do not bind JAK2 (GHR(1-318) or GH R(1-294). GH-dependent tyrosyl phosphorylation of Stat5, but not Stats 1 or 3, was reduced in CHO cells expressing GHR(1-454). GH-dependent tyrosyl phosphorylation of Stats 3 and 5 was severely reduced and unde tectable for Stat1 in cells expressing GHR(1-454) in which tyrosines 3 33 and 338 (the only tyrosines phosphorylated within 1-454) are mutate d to phenylalanine (GHR(1-454)Y333, 338F). However, GH-dependent phosp horylation of Stats 1, 3, and 5 was observed in cells expressing full- length GHR in which tyrosines 333 and 338 are mutated to phenylalanine (GHR(1-638)Y333, 338F). GH, whose receptor lacks previously defined S tat1- or Stat3-binding sites, was found in 3T3-F442A fibroblasts and 2 fTGH-GHR cells to stimulate tyrosyl phosphorylation of JAK2 to a subst antially greater extent than, and JAK1 to a similar extent as, leukemi a inhibitory factor (LIF) and/or interferon gamma (IFM-gamma), ligands whose receptors contain Stat3- and Stat1-binding sites and activate S tat3 and Stat1, respectively, better than GH. These findings suggest t hat: 1) JAK2 is required for GH-dependent phosphorylation of Stats 1, 3, and 5; 2) tyrosines 333 and/or 338 are required for maximal tyrosyl phosphorylation of Stats 1, 3, and 5; 3) Stat5 binds to a phosphoryla ted tyrosine(s) within amino acids 454-638 in addition to tyrosines 33 3 and/or 338; 4) GH stimulates tyrosyl phosphorylation of JAK1 in addi tion to JAK2 with JAK2 having a much greater response; 5) some Stat3 a nd Stat5 (and possibly Stat1) may bind to nonphosphorylated amino acid s in GHR or to phosphorylated tyrosines in proteins that bind to GHR ( e.g. JAK2) to be maximally activated; and 6) if JAK2, which contains S tat3-binding motifs, does serve as a docking site for some Stat protei ns, Stat-JAK2 binding is likely to be more important for GH than LIF o r IFN gamma in 3T3-F442A cells since GH induces 15 times more tyrosyl- phosphorylated JAK2 than LIF or IFN gamma.