Here we have identified a novel subfamily of dok multiadapter proteins, dok-4 and dok-5, which are putative links with downstream effectors of c-Ret signaling. Yeast two-hybrid analysis and coimmunoprecipitation demonstrated that dok family members, through their PTB domains, bind directly to phosphorylated tyrosyl 1062 of Ret and become phosphorylated themselves. Interaction of doks and c-Ret could also be demonstrated in tissues of the spinal cord and dorsal root ganglia. Many other receptors, such as Met, Kit, Fms, Ros, TrkA, and ErbB-2, do not interact directly with the doks. Dok-4 and dok-5 are coexpressed with c-Ret in neuronal tissues. Moreover, PC12 cells that express c-Ret/dok-4 and c-Ret/dok-5 hybrids produce neurite outgrowth upon stimulation by ligands. These data suggest that the novel doks can mediate signals required for neuronal differentiation.
The expression of the new dok family members overlap with c-Ret in tissues of the central and peripheral nervous system. A function of c-Ret signaling in the nervous system has been demonstrated by genetic experiments. For instance, the number of sensory neurons of dorsal root ganglia and motor neurons of the spinal cord is reduced in GDNF-deficient mice (Moore et al., 1996
; Sanchez et al., 1996
). Ablation of neurturin, another ligand of c-Ret, leads to loss of cells in dorsal root and trigeminal sensory ganglia (Heuckeroth et al., 1999
). In addition, it has been shown that GDNF and c-Ret play an important role in development of the enteric and sympathetic nervous system and the kidney (Schuchardt et al., 1994
; Moore et al., 1996
; Pichel et al., 1996
; Sanchez et al., 1996
). However, in the latter tissues, expression of dok family members does not overlap with that of c-Ret. However, it is possible that additional dok family members that are expressed at these sites exist, or that other adapters take over dok functions. Thus, we suggest that the newly identified dok proteins, dok-4 and dok-5, can mediate c-Ret signals in a subset of neuronal tissues. Dok family members are also expressed in other tissues where c-Ret expression is weak or has not been described (Pachnis et al., 1993
; Avantaggiato et al., 1994
). For instance, dok-4 is strongly expressed in the vascular endothelium. We found that dok-4 can also associate with the endothelial Tie-2 receptor, suggesting that dok-4 may function as a substrate for Tie-2 in endothelia. Tie-2 has already been reported to associate with Dok-2 (Jones and Dumont, 1998
); however, endothelial expression of dok-2 is not pronounced and we have not been able to detect expression in this cell type.
Other members of the dok family, dok-1–3, are mainly expressed in hematopoietic tissues. Several recent reports suggest an involvement of these dok proteins in lymphoid signaling: p62dok and dok-2 are strongly tyrosine phosphorylated in Bcr-Abl–transformed myelogenous leukemia cells (Carpino et al., 1997
; Yamanashi and Baltimore, 1997
; Di Cristofano et al., 1998
). Dok-2 (dok-R/FRIP) also binds directly to the IL-4 receptor (Nelms et al., 1998
). It is also possible that hematopoietic dok proteins act as c-Ret substrates in lymphoid cells, since recent reports suggest an involvement of c-Ret in hematopoietic differentiation (Wasserman et al., 1995
; Gattei et al., 1997
; Nakayama et al., 1999
). Phoshorylation of p62dok after c-Ret activation can also occur in a phosphotidyl inositol 3 kinase–dependent manner (Murakami et al., 1999
Dok family members have the typical features of multiadapter proteins such as membrane localization sequence (PH domain), receptor interaction domain (PTB domain), and several putative binding sites for downstream substrates (P-tyr and PXXP elements). The importance of direct association of particular substrates with specific receptor tyrosine kinases for activation of various signaling pathways has been demonstrated recently, e.g., IRSs are essential for insulin receptor function, FRS2 is important for fibroblast growth factor receptor and Trk signaling, and Gab-1 is an essential substrate for c-Met (Sun et al., 1991
; Weidner et al., 1996
; Kouhara et al., 1997
; Sachs et al., 2000
; Schaeper et al., 2000
). c-Ret can directly associate with all dok family proteins. However, dok family members have distinct expression patterns. Differential expression of these adapter proteins thus adds another layer to the complexity and specificity of signal transduction by receptor tyrosine kinases.
Members of the hematopoietically expressed doks, dok-1 and dok-2, contain long COOH-terminal sequences with many tyrosyls and PXXP motifs adjacent to the PTB domain (e.g., 10 for dok-1). These doks have been shown to associate with rasGAP, c-Abl, and Nck (Holland et al., 1997
; Yamanashi and Baltimore, 1997
). Several recent reports suggest a negative role of these dok proteins in the regulation of MAP kinase (Nelms et al., 1998
; Jones and Dumont, 1999
; Noguchi et al., 1999
; Yamanashi et al., 2000
). The dok-4/5 proteins newly identified here contain short COOH-terminal tails with fewer tyrosyls and few or no PXXP motifs, and do not bind rasGAP or Nck. Moreover, when dok-4 and dok-5 are fused to c-Ret, they strongly induce MAP kinase and Elk-1 transactivation and trigger axonal outgrowth. Thus, the two subfamilies of the dok proteins, dok-1–3 and dok-4/5, appear to take over opposite signaling functions in cells.