Joyce A. VanderKuur

Growth Hormone-dependent Phosphorylation of Tyrosine 333 and/or 338 of the Growth Hormone Receptor

Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine kinase activity, GH has recently been shown to promote the association of its receptor with JAK2 tyrosine kinase, to activate JAK2, and to promote the tyrosyl phosphorylation of both GH receptor (GHR) and JAK2. In this work, we examined whether tyrosines 333 and/or 338 in GHR are phosphorylated by JAK2 in response to GH. Tyrosines 333 and 338 in rat full-length (GHR1-638) and truncated (GHR1-454) receptor were replaced with phenylalanines and the mutated GHRs expressed in Chinese hamster ovary cells. These substitutions caused a loss of GH-dependent tyrosyl phosphorylation of truncated receptor and a reduction of GH-dependent phosphorylation of the full-length receptor. Consistent with Tyr333 and/or Tyr338 serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GH•GHR•JAK2 complexes. The Tyr to Phe substitutions did not substantially alter GH-dependent JAK2 association with GHR or tyrosyl phosphorylation of JAK2. These results suggest that Tyr333 and/or Tyr338 in GHR are phosphorylated in response to GH and may therefore serve as binding sites for SH2 domain-containing proteins in GH signal transduction pathways.

Effect of A-ring isomers of estradiol-17β on gene products in MCF-7 cells

Abstract Two-dimensional polyacrylamide gel-electrophoresis analysis of in vitro translation products from extracted cellular mRNAs was utilized to examine the effect of A-ring isomers of estradiol (E 2 ) on the synthesis of proteins involved in the response of MCF-7 cells to estrogens. An 8 h pulse with 10 −8 M E 2 showed 11 polypeptides of interest, 9 displayed a transitent increase in mRNA accumulation and 2 showed a temporary decreased level in the presence of this hormone. A distinct set of 2 mRNAs displayed increased amounts only after a 24 h E 2 pulse. Position of the A-ring hydroxyl group on the estratrien-17β-ol moiety had a discriminatory effect on the mRNAs for the 11 polypeptides responsive to E 2 . The accumulation of three mRNAs (A, C, and E) were increased by the 3 A-ring isomers (1-, 2-, and 4-hydroxyestratrien-17β-ol) to a degree comparable to that brought about by E 2 . One mRNA (H) was decreased by all estrogens. The pattern of responses depicted in the remaining 7 polypeptides was different depending on the position of the A-ring hydroxyl group of the estrogen. Subtle changes in the structure of E 2 appear to attenuate the ability of this natural ligand to regulate certain estrogen responsive genes and not others. This phenomenon may be related to the interaction of TAF-2 in ligand bound receptor with the various regulators in the promoter region of specific estrogen responsive genes. (Steroids 59 :548–554, 1994)