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Fig 5. XRnf12 causes ubiquitination and proteasome-dependent degradation of Ldb1 in a RING-dependent manner. (A) XRnf12 does not affect the steady-state level of Xlim-1-FLAG, either in the presence or absence of Ldb1. The indicated mRNAs were injected into the ventral region and the expression levels of the FLAG-tagged proteins were examined at the gastrula stage. Note that the levels of Xlim-1-FLAG are increased in the presence of Ldb1. β-tubulin, loading control. Amounts of mRNAs injected (ng/embryo): Xlim-1-FLAG, 0.25; Ldb1, 0.5; XRnf12, 0.25. (B) XRnf12 decreases the steady-state level of FLAG-Ldb1 both in the presence and absence of Xlim-1 in a RING-dependent manner. Note the increase in the expression level of FLAG-Ldb1 by Xlim-1 co-expression. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1, 0.25; XRnf12 constructs, 0.25. (C) XRnf12 enhances ubiquitination of Ldb1. Embryos were injected ventrally with the mRNAs indicated and the cell lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by either anti-FLAG or anti-HA immunoblotting (IB) to detect non-ubiquitinated Ldb1 or ubiquitinated proteins, respectively. Co-expression of XRnf12 results in downregulation of non-ubiquitinated FLAG-Ldb1 expression levels (lower panel). While weak ubiquitination is observed in the absence of XRnf12 (lane 4), strong ladder-like ubiquitination signals appear in the presence of XRnf12 (lane 7). XRnf12(HC>AA) does not enhance ubiquitination (lane 8). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 2.0; HA-Ub, 1.0; XRnf12 constructs, 1.0. (D) The N-terminal region (aa 1-291) of Ldb1 is sufficient for ubiquitination by XRnf12. By using FLAG-Ldb1ΔC instead of FLAG-Ldb1, smaller-sized ubiquitinated protein bands are detected, confirming that the ubiquitinated proteins in C are indeed Ldb1 and not some other proteins associated with Ldb1. Co-expression of XRnf12 also results in downregulation of non-ubiquitinated FLAG-Ldb1ΔC. The amounts of mRNAs used are the same as in C. Arrowhead indicates the position of IgG. (E) XRnf12 causes proteasome-dependent degradation of Ldb1. After mRNA injection, cells were dispersed and cultured in the presence or absence of MG-132 until the gastrula stage. Decrease of FLAG-Ldb1 levels by XRnf12 (lane 5) is suppressed in the presence of MG-132 (lane 6). MG-132 does not affect the expression of FLAG-Ldb1 (lanes 3,4). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1, 0.25; XRnf12, 0.25. (F) The steady-state level of FLAG-Ldb1 is downregulated by hRNF6 and, to a lesser extent, by hRNF38, but not by hRNF13. The experimental design is the same as in A and B. β-tubulin, loading control. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; RING finger proteins, 0.5. (G) RING finger proteins that cause reduction in the steady-state level of Ldb1 interact with Ldb1. GST pull-down assay was performed with 35S-labeled XRnf12, hRNF6, hRNF13 and hRNF38. Human RNF13 does not interact with GST-Ldb1, while other RING finger proteins do. GST serves as a negative control. Image published in: Hiratani I et al. (2003) Copyright © 2003. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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