Activation of Stat3 in v-Src-transformed Fibroblasts Requires Cooperation of Jak1 Kinase Activity

Yi Zhang,James Turkson,Christin Carter-Su,Thomas Smithgall,Alexander Levitzki,Alan Kraker,John J Krolewski,Peter Medveczky,Richard Jove

doi:10.1074/jbc.m002383200

Abstract

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon activation by tyrosine phosphorylation. Several protein-tyrosine kinases can induce phosphorylation of STATs in cells, including Janus kinase (JAK) and Src family kinases. One STAT family member, Stat3, is constitutively activated in Src-transformed NIH3T3 cells and is required for cell transformation. However, it is not entirely clear whether Src kinase can phosphorylate Stat3 directly or through another pathway, such as JAK family kinases. To address this question, we investigated the phosphorylation of STATs in baculovirus-infected Sf-9 insect cells in the presence of Src. Our results show that Src can tyrosine-phosphorylate Stat1 and Stat3 but not Stat5 in this system. The phosphorylated Stat1 and Stat3 proteins are functionally activated, as measured by their abilities to specifically bind DNA oligonucleotide probes. In addition, the JAK family member Jak1 efficiently phosphorylates Stat1 but not Stat3 in Sf-9 cells. By contrast, we observe that AG490, a JAK family-selective inhibitor, and dominant negative Jak1 protein can significantly inhibit Stat3-induced DNA binding activity as well as Stat3-mediated gene activation in NIH3T3 cells. Furthermore, wild-type or kinase-inactive platelet-derived growth factor receptor enhances Stat3 activation by v-Src, consistent with the receptor serving a scaffolding function for recruitment and activation of Stat3. Our results demonstrate that Src kinase is capable of activating STATs in Sf-9 insect cells without expression of JAK family members; however, Jak1 and platelet-derived growth factor receptor are required for maximal Stat3 activation by Src kinase in mammalian cells. Based on these findings, we propose a model in which Jak1 serves to recruit Stat3 to a receptor complex with Src kinase, which in turn directly phosphorylates and activates Stat3 in Src-transformed fibroblasts.

Highlights

J To whom correspondence should be addressed: Molecular Oncology Program, Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, FL 33612
Our results demonstrate that Src kinase is capable of activating STATs in Sf-9 insect cells without expression of Janus kinase (JAK) family members; Jak1 and platelet-derived growth factor receptor are required for maximal Stat3 activation by Src kinase in mammalian cells
Src is capable of directly activating Stat3, we investigated if the mechanism of Stat3 activation is through direct interaction with Src kinase independent of other protein-tyrosine kinases, such as JAK

Summary

Introduction

J To whom correspondence should be addressed: Molecular Oncology Program, Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, FL 33612. While receptor tyrosine kinases may directly phosphorylate STATs, some reports suggest that JAKs are involved in PDGF-induced STAT activation. We examined whether inhibition of JAK kinases in vivo will affect Stat3 DNA-binding activity in fibroblasts stably transformed v-Src. NIH3T3 cells transformed with v-Src were treated in vivo with various concentrations of AG490 or PD180970.

Results

Conclusion