Src-CrkII-C3G-dependent Activation of Rap1 Switches Growth Hormone-stimulated p44/42 MAP Kinase and JNK/SAPK Activities

Ling Ling,Tao Zhu,Peter E Lobie

doi:10.1074/jbc.m302516200

Abstract

We demonstrate here that growth hormone (GH) stimulates the activation of Rap1 and Rap2 in NIH-3T3 cells. Full activation of Rap1 and Rap2 by GH necessitated the combined activity of both JAK2 and c-Src kinases, although c-Src was predominantly required. GH-stimulated Rap1 and Rap2 activity was also demonstrated to be CrkII-C3G-dependent. GH stimulated the tyrosine phosphorylation of C3G, which again required the combined activity of JAK2 and c-Src. C3G tyrosine residue 504 was required for GH-stimulated Rap activation. Activated Rap1 inhibited GH-stimulated activation of RalA and subsequent GH-stimulated p44/42 MAP kinase activity and Elk-1-mediated transcription. In addition, we demonstrated that C3G-Rap1 mediated CrkII enhancement of GH-stimulated JNK/SAPK activity. We have therefore identified a linear JAK2-independent pathway switching GH-stimulated p44/42 MAP kinase and JNK/SAPK activities.

Highlights

The Rap proteins (Rap1 and Rap2) belong to the Ras-like small GTPase superfamily, which consists of at least 13 members [1]
growth hormone (GH) is the primary regulator of postnatal somatic growth and metabolism [18, 19]. It utilizes special groups of signaling molecules to regulate the transcription of specific genes required for the above processes. These signaling molecules include: 1) receptor-tyrosine kinases (EGF receptor) [20] and non-receptor-tyrosine kinases (JAK2, Ref. 3; c-Src; c-Fyn, Ref. 21; and FAK, Ref. 22), in the case of the EGF receptor it may be used as an adaptor protein; 2) members of the MAP kinase family including p44/42 MAP kinase [23], p38 MAP kinase [24], and JNK/SAPK [21] and their respective downstream effectors; 3) members of the insulin receptor substrate (IRS) group including IRS-1, -2, and -3, which may act as docking proteins for further activation of signaling molecules including phosphatidylinositol 3-kinase [25]; 4) small Ras-like GTPases [26]; and 5) STAT family members including STATs 1, 3, 5a, and 5b [27, 28], which constitute one group of signaling molecules involved in transcriptional regulation by GH
We have demonstrated that cellular stimulation with GH results in the activation of both Rap1 and Rap2 in NIH-3T3 cells

Summary

GH Activates Rap

Vation of p44/42 MAP kinase by GH in a c-Src-dependent manner. We have demonstrated that cellular stimulation with GH results in the activation of both Rap and Rap in NIH-3T3 cells. The activation of Rap by GH was achieved by the combined JAK2- and c-Src-dependent tyrosine phosphorylation of C3G. GH-stimulated Rap activation was utilized to negatively modulate GH-stimulated p44/42 MAP kinase activity and subsequent Elk-1-mediated transcription through inhibition of RalA activity. GH stimulated C3G-dependent activation of Rap1-enhanced JNK/SAPK activity and subsequent c-Jun-mediated transcription in response to GH. Rap is a GH effector molecule activated in a JAK2-independent manner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION