Wnt signaling plays crucial roles in cell proliferation, migration and morphogenetic movements that are important in both embryonic development and cancer progression. Here, we report a new role of Wnt-8 and CKIε in the regulation of a small GTPase, Rap1. The data reveals dual functions of CKIε in Wnt signaling. CKIε both regulates gastrulation by modulating Rap1 pathway, and activates Lef1-TCF dependent gene expression by stabilizing β-catenin ().
Although previous studies of CKIε in Xenopus
development suggested it functions in non-canonical Wnt signaling, details of the downstream signal transduction pathways have been unclear (McKay et al., 2001
). Non-canonical Wnt pathways affect cell movements rather than gene transcription, making them more difficult to assay. Second, single time-point assays in cell-based systems tend to miss dynamic effects of morphological progression. Here, we first utilized cell-based systems to determine how CKIε regulates the SIPA1L1-Rap1 pathway, and then tested the biological effects in Xenopus
and zebrafish development. The data suggests CKIε is involved in multiple pathways downstream of Wnt. Prior to gastrulation, CKIε enhances β-catenin dependent gene expression, thus controlling cell fate specification (). However, during gastrulation, CKIε regulates convergent extension through the SIPA1L1-Rap1 pathway. Consistent with this, DN-Rap1 or CKIε-resistant SIPA1L1 (SIPA1L1(CΔ)) do not affect β-catenin-dependent gene expression in early gastrula stages, but inhibit convergent extension during gastrula and neurula stages of Xenopus
development (, and ).
Wnt-8 has been classified as a “canonical” Wnt because it can induce axis duplication in Xenopus
embryos and β-catenin dependent gene expression (Christian and Moon, 1993
). However, classifying Wnts by their ability to induce axis duplication or to activate a TCF-dependent promoter may be imprecise, since pathway activation also depends on receptor expression in target cells and cross-talk between canonical and non-canonical signaling has been reported (Kimura-Yoshida et al., 2005
; Tao et al., 2005
). Our data indicates a role for Wnt-8 in a non-canonical Wnt pathway. Consistent with previous studies that regulation of convergent extension does not require β-catenin, we found that β-catenin could not moderate the CE defect caused by downregulation of Rap1. We suggest that the CKIε/β-catenin and CKIε/RAP1 pathways together contribute to proper axis development. During axis development, Wnt-8 (and others) activates regional β-catenin distribution, initiating axis formation, and later activates the Rap1 pathway, facilitating morphogenic movements.
Rap GTPases include Rap1 and Rap2, which share about 60% amino acid identity. While both are involved in cell migration and adhesion, their functions in Xenopus
development appear to be distinct. Although Rap2 affects β-catenin mediated dorsalization events (Choi and Han, 2005
), we find that Rap1 is involved in a non-canonical Wnt pathway. Unlike Rap2, Rap1 knockdown causes a defect in gastrulation rather than a hyper-ventralized phenotype (). Inhibition of Rap1 does not change the expression of Gsc
), suggesting that Rap1 signaling does not affect dorsal-ventral specification. Two prior observations are also consistent with the conclusion that a SIPA1L1-Rap1 pathway regulates morphogenetic movements in embryogenesis. First, Rap1 regulates cell migration in cell-based studies (McLeod et al., 2002
), consistent with a role in morphological movements during development. Second, SIPA1L1 induces actin reorganization in rat neurons via its GAP activity (Pak et al., 2001
). Although SIPA1L1 can facilitate GTP hydrolysis both in Rap1 and Rap2 (Singh et al., 2003
), SIPA1L1 expression disrupts gastrulation rather than inducing a hyper-ventralized phenotype. SIPA1L1 may therefore preferentially affects Rap1 during development.
Two small GTPases, Rho and Rac, are known Wnt-regulated small GTPase required for proper gastrulation during Xenopus
development (Habas et al., 2003
; Habas et al., 2001
). Biochemical analysis of Rho and Rac regulation indicates that Wnt signaling activates these pathways through parallel mechanisms (Habas et al., 2003
). Both distinct and overlapping functions of Rho and Rac are essential for directing CE (Tahinci and Symes, 2003
). Our study identifies Rap1 as an additional small GTPase downstream of Wnt. Rap1, Rho, and Rac may be cooperatively activated by a Wnt signal to mediate the complex series of movements involved in gastrulation. Rap1 has previously been found to be activated by changes in cytoskeletal tension (Tamada et al., 2004
). We speculate that Rap1 activity facilitates changes in cell-cell adhesion required for proper morphogenetic movements.
Wnt signaling, via CKIε and SIPA1L1, regulates Rap1, and Rap1 plays an essential role in embryogenesis. SIPA1L1/E6TP1 is also a target of the oncogenic human papillomavirus protein E6, suggesting its inactivation contributes to cancer development (Gao et al., 2001
). E6-mediated SIPA1L1/E6TP1 degradation correlates with the immortalization of epithelial cells (Gao et al., 2001
). In addition, mutations of SIPA1, a SIPA1L1 related protein, correlates with efficiency of breast cancer metastasis (Crawford et al., 2006
; Park et al., 2005
). The fact that Wnt signaling and CKIε activity also decrease SIPA1L1, combined with the fact that some Wnts are oncogenes, is consistent with the hypothesis that SIPA1L1 downregulation contributes to malignant transformation. The role of Rap1 in cell migration suggests Wnt signaling may therefore play a role not only in proliferation but in tumor invasion and metastasis as well.