|M.Sc Student||Adi Ayala|
|Subject||Signaling Mechanisms of Growth Hormone in Human Prostate|
|Department||Department of Medicine||Supervisor||Assistant Professor Ronnie Barkey|
This thesis demonstrates initial functional responses to GH in LNCaP cells, based on the study of GH-induced signaling. Western analysis of lysates showed that a 120 kDa protein, identified as Janus tyrosine kinase (JAK2), was phosphorylated in a time- and dose-dependent manner. GHR appeared as a broad band of 111-120 kDa. JAK2 appears to be constitutively associated to GHR. GH also induced phosphorylation of a 97 kDa protein, identified as signal transducer and activator of transcription (STAT5a). No phosphorylation of 93 kDa STAT5b was detected. GH is also known to activate the mitogen-activated protein kinase (MAPK; Erk1/2) in some cell types. Erk1/2 were phosphorylated in a dose- and time-dependent manner. The PI3K plays an important role in proliferation and survival of cells by activating protein kinase B (PKB/AKT). AKT was shown in LNCaP cells, as a 60 kDa protein, which was phosphorylated in a dose- and time-dependent manner following exposure to hGH.
In order to demonstrate the key role of JAK2 as the initial activation step in the signal transduction of hGH, we made use of a JAK2-specific inhibitor, AG-490. Exposure of LNCaP cells to AG-490 reduced hGH-induced JAK2 phosphorylation by up to 50%. AG-490 reduced by up to 65% the phosphorylation of Erk1/2 and reduced by up to 80% the hGH-induced phosphorylation of AKT. In order to demonstrate the possibility of cross-talk between the PI3K pathway, the activation of its substrate, AKT, and the phosphorylation of Erk1/2, we made use of a PI3K-inhibitor, wortmannin. Exposure of LNCaP cells to wortmannin prevented the hGH-induced phosphorylation of AKT and reduced basal AKT phosphorylation by up to 95%. Wortmannin also caused a reduction of up to 75% in the Erk1/2 phosphorylation. In contrast, the protein kinase C (PKC) inhibitor GF109203x had no effect on the hGH-induced Erk1/2 activation, ruling out the involvement of the PKC pathway in the activation of Erk1/2 in response to hGH.