STAT activation by the PDGF receptor requires juxtamembrane phosphorylation sites but not Src tyrosine kinase activation Christoph Sachsenmaier 1 , Henry B Sadowski 2,3 and Jonathan A Cooper* ,1 1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA Activation of the platelet-derived growth factor (PDGF) receptor tyrosine kinase induces tyrosine phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. Since the PDGF receptor also activates the Src tyrosine kinase, it is possible that Src mediates tyrosine phosphorylation of STATs in PDGF- treated cells. Consistent with a role for Src in STAT activation, we found that a PDGF receptor juxtamem- brane tyrosine residue required for Src activation is necessary and sucient for activation of STATs 1 and 3. To test the Src requirement further, we made other mutations in the PDGF receptor juxtamembrane region that increased or decreased Src binding. In epithelial and ®broblast cells, PDGF activated STAT1, 3 and 6 in the absence of detectable binding and activation of Src. In addition, PDGF induced c-myc RNA expression and DNA synthesis even though Src was not detectably activated. The activation of MAP kinase and the induction of c-fos gene expression both correlated with STAT but not Src activation by the receptor. We conclude that juxtamembrane tyrosine phosphorylation is necessary for both Src tyrosine kinase and STAT activation by the bPDGF receptor, but that both processes are regulated independently by this region. Keywords: Src; STAT; PDGF; receptor tyrosine kinase; juxtamembrane Introduction Members of the Src family of tyrosine kinases play important roles in regulating cell growth, differentia- tion and transformation. This family comprises at least eight members in mammals that share sequence homologies with c-Src; the ®rst cellular protein tyrosine kinase and oncogene described (Brown and Cooper, 1996; Thomas and Brugge, 1997). Src is activated in response to various external stimuli and by various mechanisms. In the case of PDGF-treated cells, a subset of Src molecules is recruited to activated bPDGF receptors by binding of the Src SH2 domain to phosphorylated tyrosine residues within the juxtamembrane domain of the receptor (Mori et al., 1993). This binding is thought to activate Src by displacing an intramolecular interaction between the Src SH2 domain and a C-terminal phosphorylated tyrosine residue (Cooper and Howell 1993). Total cellular Src activity rises transiently 2 ± 4-fold within minutes after stimulation of resting cells with PDGF (Ralston and Bishop, 1985; Gould and Hunter, 1988). The initial binding and activation of Src family members to the bPDGF receptor appears to be necessary for PDGF-induced DNA synthesis and expression of the c-myc gene (Twamley-Stein et al., 1993; Barone and Courtneidge, 1995). Src family members may also regulate expression of genes through phosphorylation and activation of transcrip- tion factors known as STATs. Recently it was shown that an activated mutant of Src, v-Src, constitutively phosphorylates and activates STAT3, and that STAT3 phosphorylation is necessary for ecient transforma- tion of cells by v-Src (Yu et al., 1995; Cao et al., 1996; Chaturvedi et al., 1997; Bromberg et al., 1998; Turkson et al., 1998). The phosphorylation of STAT3 by v-Src suggests that normal cellular Src may also phosphory- late STAT3 and directly regulate transcription. The family of STAT proteins was identi®ed during studies of cytokine-induced gene expression (Darnell, 1997). Cytokine receptors lack intrinsic tyrosine kinase activity but are associated with cytoplasmic tyrosine kinases of the JAK family (Ihle and Kerr, 1995). Upon cytokine receptor oligomerization, JAKs phosphorylate the receptor molecules on tyrosine. This allows the receptor to bind STATs through their SH2 domains (Heim et al., 1995; Stahl et al., 1995). STATs are then phosphorylated by JAKs on a conserved tyrosine residue, which drives the subsequent events of STAT release, dimerization and nuclear translocation (Briscoe et al., 1996). Alternative mechanisms of STAT activation by JAKs which involve JAK/STAT interac- tion without a cytokine receptor scaold have also been described (Fujitani et al., 1997). Growth factors that act through receptors with intrinsic tyrosine kinase activity also activate STAT family members. Less is known about the role of STATs in growth factor signaling than in cytokine signaling, but both growth stimulatory and inhibitory pathways are stimulated (Wagner et al., 1990; Chin et al., 1996; Su et al., 1997). The mechanism of tyrosine phosphorylation of STATs in growth factor treated cells is also unclear. The tyrosine kinase activities of the PDGF and EGF receptor tyrosine kinases are required for activation of STATs by PDGF and EGF, respectively, but it is not known whether these receptors directly phosphorylate STAT proteins or do so via another protein tyrosine kinase(s). Indeed, JAK family members become activated in growth factor *Correspondence: JA Cooper, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North ± Mailstop A2-025, P.O. Box 19024, Seattle, Washington 98109-1024, USA Current address: 3 Department of Biochemistry, Mount Sinai Medical Center, New York, New York 10029, USA Received 24 August 1998; revised 14 January 1999; accepted 14 January 1999 Oncogene (1999) 18, 3583 ± 3592 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc