SHORT COMMUNICATION The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase B Linggi, QC Cheng, AR Rao and G Carpenter Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA The ErbB-4 receptor tyrosine kinase homo- and hetero- dimerizes following heregulin binding, which provokes increased levels of tyrosine autophosphorylation. Unique totheErbBfamily,ErbB-4isthenproteolyticallycleaved by a- and c-secretase to produce an 80kDa intracellular domain (s80 ICD) fragment. This fragment is found in both the cytoplasm and nucleus of many normal and cancer cells and can interact with transcription factors in the cytoplasm and nucleus. Since the s80 ICD lacks ectodomain sequences known to play a major role in dimerization of ErbB family members, we asked whether the s80 ICD is an active tyrosine kinase. Here, we demonstrate that the s80 ICD is a constitutively active tyrosinekinaseandcanformhomodimers.Thes80ICDis autophosphorylated in cells and can phosphorylate an exogenous substrate in vitro. Also, the s80 ICD can coassociate and dimers are detected by chemical cross- linking. This is the first example of constitutive kinase activation and dimerization totally within the cytoplasmic domain of an ErbB receptor and suggests that the s80 ICD may function to phosphorylate substrates in the cytoplasm or nucleus. Oncogene (2006) 25, 160–163. doi:10.1038/sj.onc.1209003; published online 19 September 2005 Keywords: ErbB-4; tyrosine kinase; growth factor ErbB-4 is a member of the ErbB receptor tyrosine kinase family, which also includes ErbB-1 (EGF receptor), ErbB-2 and ErbB-3 (Carpenter, 2003). In the cases of ErbB-1 and ErbB-4, ligand binding provokes receptor homodimerization and heterodimerization with ErbB-2. Both homo- and heterodimers represent productive signaling complexes (Yarden and Sliwkowski, 2001). ErbB-2 does not bind a known ligand and functions primarily within heterodimers to modify post-receptor signaling events. In each instance, growth factor- induced dimerization leads to activation of tyrosine kinase activity intrinsic to the cytoplasmic domains of ErbB-1, ErbB-2 and ErbB-4. Recent crystallographic studies of ErbB ectodomains have illuminated the role of an ectodomain dimerization loop as a required element in growth factor initiated dimerization of ErbB family members (Burgess et al., 2003). However, these results do not allow the conclu- sion that exposure of the ectodomain dimerization loop is sufficient for dimerization of the intact receptor kinase. There is evidence that sequences within the transmembrane domain and/or cytoplasmic domains may also be involved in the dimerization mechanism of the intact receptors (Chantry, 1995; Murali et al., 1996; Penuel et al., 2002; Stamos et al., 2002; Zhu et al., 2003; Landau et al., 2004; Aifa et al., 2005). Within this receptor family only ErbB-4 is subject to a novel two-step proteolysis pathway that is stimulated by binding of the cognate growth factor, heregulin (neur- egulin), or the addition of TPA (Vecchi et al., 1996; Zhou and Carpenter, 2000; Ni et al., 2001; Lee et al., 2002). The initial cleavage occurs within the ErbB-4 ectodomain between His651 and Ser652, placing this cleavage site eight residues outside the transmembrane domain (Cheng et al., 2003). It seems likely that this cleavage is executed by the transmembrane metallopro- tease TACE (ADAM17), as TACE null cells fail to initiate TPA-provoked cleavage (Rio et al., 2000) and recombinant TACE cleaves a peptide representing ErbB-4 residues 646–657 between His651 and Ser652 (Cheng et al., 2003). In this system, TACE functions as an a-secretase. The products of ErbB-4 ectodomain cleavage are an ectodomain fragment of 120 kDa and membrane-asso- ciated 80kDa fragment (m80) that begins with Ser652 and includes the transmembrane and cytoplasmic domains of ErbB-4 (Vecchi et al., 1996; Cheng et al., 2003). The m80 fragment serves as substrate for a second cleavage event that is mediated by the g-secretase component presenilin, a polytopic transmembrane protein, which cleaves substrates within the transmem- brane domain. g-Secretase converts the m80 substrate to a product that is designated s80 and is found in the cytosol and nucleus (Ni et al., 2001; Lee et al., 2002). The role of the s80 fragment, also termed the intracellular domain (ICD), in biologic responses to heregulin has been investigated recently. The results indicate that in mammary cells this s80 ICD fragment can provoke events associated with differentiation (Williams et al., 2004). This includes a chaperone function to mediate the nuclear entry of STAT5a and Received 28 April 2005; revised 8 July 2005; accepted 11 July 2005; published online 19 September 2005 Correspondence: Dr G Carpenter, Department of Biochemistry, Vanderbilt University School of Medicine, 647 Light Hall, Nashville, TN 37232-0146, USA. E-mail: graham.carpenter@vanderbilt.edu Oncogene (2006) 25, 160–163 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc