Regulation of STAT protein synthesis by c-Cbl Warren A Blesofsky 1 , Kerri Mowen 1 , Robert M Arduini 3 , Darren P Baker 3 , Maria A Murphy 2 , David DL Bowtell 2 and Michael David* ,1 1 Department of Biology and UCSD Cancer Center, University of California San Diego, La Jolla, California, USA; 2 Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Australia; 3 Biogen, Inc., Cambridge, Massachusetts, USA Many cytokines and growth factors induce transcription of immediate early response genes by activating members of the Signal Transducers and Activators of Transcrip- tion (STAT) family. Although significant progress has been made in understanding the events that lead to the activation of STAT proteins, less is known about the regulation of their expression. Here we report that murine embryonic fibroblasts derived from c-Cbl-de- ficient mice display significantly increased levels of STAT1 and STAT5 protein. In contrast, STAT2 and STAT3 expression, as well as the levels of the tyrosine kinases Jak1 and Tyk2, appear to be regulated independently of c-Cbl. Interestingly, the half-life of STAT1 was unaected by the presence of c-Cbl, indicating that c-Cbl acts independently of STAT1 degradation. Further analysis revealed similar levels of STAT1 mRNA, however, a dramatically increased rate of STAT1 protein synthesis was observed in c-Cbl- deficient cells. Thus, our findings demonstrate an additional control mechanism over STAT1 function, and also provide a novel biological eect of the Cbl protein family. Oncogene (2001) 20, 7326 – 7333. Keywords: Interferon; c-Cbl; STAT; translation Introduction Signal transducers and activators of transcription (STATs) represent a family of transcription factors that mediate cytokine and growth factor-induced activation of immediate early response genes in the absence of de-novo protein synthesis (Darnell et al., 1994; Larner et al., 1993). Seven genetically distinct mammalian STAT proteins have been described (Fu, 1992; Lin et al., 1996; Liu et al., 1995; Quelle et al., 1995; Yamamoto et al., 1994; Zhong et al., 1994a,b), and related signaling molecules have been found in Drosophila (Hou et al., 1996; Yan et al., 1996) and Dictyostelium (Kawata et al., 1997). All STAT proteins are activated through rapid tyrosine phosphorylation (Gupta et al., 1996; Heim et al., 1995), which is required for dimerization (Shuai et al., 1994), nuclear translocation (Mowen and David, 1998) and DNA binding (David et al., 1993; Fu, 1992). Tyrosine phosphorylation of STAT proteins in response to cytokines requires the activity of one or more Janus tyrosine kinases (JAKs), whereas STAT phosphoryla- tion via growth factor receptors such as the epidermal growth factor receptor depends on the intrinsic kinase activity of the receptor. In addition to phosphorylation of STAT1 and STAT3 on Tyr701 or Tyr705, respectively, further phosphorylation on Ser727 is essential to maximize their transactivation capabilities (Wen et al., 1995). Although more emphasis had been placed on STAT activation than on inactivation, several negative regulatory components of the Jak/ STAT pathway have recently been identified. These proteins target either the activating Jak kinase, or aect the phosphorylated STAT protein directly. However, in either case, it is the activation state or the functionality of the STAT proteins that is aected rather than their protein levels. The proto-oncogene c-Cbl was originally identified as the mammalian ortholog of the v-Cbl oncogene isolated from the Cas NS-1 murine leukemia virus (Blake et al., 1991; Langdon et al., 1989). Homologs of c-Cbl have been found in Drosophila (D-Cbl) (Meisner et al., 1997) and in C. elegans (sli-1) (Yoon et al., 1995). The 120 kDa cytoplasmic gene product of c-Cbl, which lacks any detectable enzymatic activity, contains in its amino-terminus a unique SH2-domain, an EF hand domain, and a highly basic region. The COOH- terminal half contains a proline-rich domain, several tyrosine phosphorylation sites, and a ubiquitin-asso- ciated (UBA) domain. A centrally located ring-finger domain has been demonstrated to be essential for c- Cbl’s function as a ubiquitination-promoting or E3 factor (Joazeiro et al., 1999). Two oncogenic forms of c-Cbl lack this critical feature. The v-Cbl protein represents a carboxy-terminal deletion, lacking the ring-finger and the proline-rich domain. 70Z-Cbl, which was isolated from a pre-B cell line, contains an internal deletion within the ring-finger domain (amino acids 366 – 382), illustrating that mutations or deletions of the ring finger domain can be responsible for the oncogenic potential of c-Cbl (Blake et al., 1991). Much of the eort in understanding the function of c-Cbl has been focused on its role in the ubiquitin- Oncogene (2001) 20, 7326 – 7333 ª 2001 Nature Publishing Group All rights reserved 0950 – 9232/01 $15.00 www.nature.com/onc *Correspondence: M David, University of California, San Diego, Department of Biology, Bonner Hall 3138, 9500 Gilman Drive, La Jolla, CA 92093-0322, USA; E-mail: midavid@ucsd.edu Received 6 July 2001; revised 7 August 2001; accepted 14 August 2001