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CKIP-1 interacts with Smurf1 both in vivo in vitro Mapping the interacting regions Neither Smurf2 nor other members of Nedd4 family interacts with CKIP-1 Paradox: binding WW domains v.s. E3-promoting effect Phospho-lipid binding Substrate binding E2 binding (N-lobe) Ubiquitin binding (C-lobe) C2 HECT WWs Two possibilities: 1, CKIP-1 and substrate bind to each of two WW domains. 2, CKIP-1 binds to the linker region between the two WW domains. Smurf1 CKIP-1 binds to the linker region between WW domains of Smurf1 The linker region is highly homologous but different between Smurf1 and Smurf2. Mapping the key amino acids in the linker A The linker is essential for binding 细节决定成败! Skp1 brings F-box protein to Cul1 Bai et al. (1996) Cell 86:263-274 Cullin 1 Skp1 F-box ROC1 E2 Ub Cullin 1 Skp1 F-box ROC1 E2 Ub substrate Nd F-box proteins: substrate receptors F-box proteins W L O CDC4 F E2 Ub Cullin 1 Roc1 Skp1 Ub Ub Ub Ub Ub Ub Ubiquitination of CDK inhibitor SIC1 by SCFCDC4-ROC ligase P P SIC1 SCF-ROC E3 ubiquitin ligase Zheng et al. (2002) Nature 416:703 SCF-ROC ligases E2 Ub Cullin 1 Roc1 Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub …… Skp1 Skp2 p27 F Skp1 Fbp 4 Substrate 4 F Skp1 Fbp 5 Substrate 5 F Skp1 Fbp 6 Substrate 6 F Skp1 Fbp 7 Substrate 7 F Skp1 Fbp 63 Substrate 50 F b-TrcP IkBa F Fbp 3 Substrate 3 F Human Cullins and Cullin-related proteins Kipreos et al. (1996) Cell 85:829 C U L 5 7 8 0 A P C 2 8 2 2 N d 8 C U L 1 S K P 1 7 7 6 C U L 2 7 4 5 C U L 3 7 6 8 C U L 4 A 7 5 9 C U L 4 B 7 1 7 ? ? ? ? ? ? R O C Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub BCR (BTB-CUL3-ROC) E3 ligases Cullin 3 Roc1 E2 Ub Ub Ub Ub Ub Ub Ub …… BTB4 Substrate 4 BTB BTB5 Substrate 5 BTB BTB6 Substrate 6 BTB BTB5 Substrate 5 BTB BTB6 Substrate 6 BTB BTB200 Substrate 200 BTB MEI-1 MEL26 BTB NRF2 Keap1 BTB Substrate3 BTB3 BTB Cullin4: DDB1-DCAF-ROC ligases E2 Ub Cullin 4 Roc1 Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub …… DDB1 DDB2 XPC WD DDB1
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