SUPPLEMENTARY MATERIAL
Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd42 using proteome arrays
Avinash Persaud, Philipp Alberts, Eva M. Amsen, Xuejian Xiong, James Wasmuth, Zachary Saadon, Chris Fladd, John Parkinson and Daniela Rotin*
Programs in Cell Biology and Molecular Structure and Function, The Hospital for Sick Children, and Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
*Correspondence: Dr. Daniela Rotin The Hospital for Sick Children TMDT-MaRS, Rm 305 101 College St., Toronto, Ontario, Canada, M5G 1L7 Tel: (416) 813-5098 FAX: (416) 813-8456 Email:
[email protected]
Supplementary Figure S1: Ubiquitylation assays on human protoarrays probed with rNedd4-1, hNedd4-1 and hNedd4-2 (details are provided in the legend to Figure 3 and the Materials and Methods).
Supplementary Figure S2: Binding assays on human protoarrays probed with rNedd4-1, hNedd4-1 and hNedd4-2 (details are provided in the legend to Figure 3 and the Materials and Methods).
Supplementary Figure S3: The RxxQE motif does not serve as recognition site for Nedd4 proteins. HEK293T cells were co-transfected (or not) with the cDNA for wildtype (WT) CACNB1 or ANXA9, or their mutants versions with the RxxQE sequence mutated to Ala (AxxAA, called RxxQE-mut), along with the corresponding Nedd4 protein (WT or catalytically-inactive CS mutant), and binding (A) or ubiquitylation (B) assays performed as described in the legend to Fig 5 and in the Methods section. Lysate mixing (LM) in panel (A) signifies mixing of lysates after cell lysis, to ensure that the binding seen in the other lanes occurred inside the cells and not after the cells were lysed. The two left lanes in both blots in panel A depict positive and negative controls for Nedd4 binding: LAPTM5 and tits PY-motif mutant, respectively. LAPTM5 was previously shown to bind Nedd4 via its PY motifs.
Supplementary Figure S4. Conservation of Nedd4-1 and Nedd4-2 substrates (A) Frequency of four classes of human genes with orthologs across 111 eukaryotic genomes. "Nedd4 substrates" represent the 111 "high confidence" substrates identified in this study. "Translation" represent 724 genes annotated with the Gene Ontology term "translation" (GO:0006412). "Signaling" represent 2036 genes annotated with the Gene Ontology term "Signal transduction" (GO:0006412). Orthologs were determined using the InParanoid algorithm. (B) Conservation profiles of 111 Nedd4 substrates (vertical axis) compared to 111 eukaryotic genomes (horizontal axis). Each coloured tile indicates the type of orthologous relationship of the Nedd4 substrate to the comparator genome (see inset key for details). The genomes are grouped and ordered on the basis of known phylogenetic relationships (see Suppl. Table SIII for further details). Genes were clustered on the basis of Euclidean distance using complete linkage. The yellow outline indicates the presence of putative yeast orthologs. Colored backgrounds on the Nedd4 substrate genes indicate: blue = substrate of hNedd4-1 or rNedd4-1; red = substrate of hNedd4-2; purple = substrate of hNedd4-1 and/or rNedd4-1 and hNedd4-2.
Supplementary Figure S5: Sustained EGF-mediated signalling in Nedd4-1-/- MEFs Wildtype (WT) and Nedd4-1-/- knockout (KO) MEFs were incubated without (starved- time 0) or with EGF for 5, 30, 60 or 120 min. EGF-dependent signalling was tested by immunoblotting with phosphospecific antibodies to Erk (pErk) and compared to total expression of Erk. Lower panels: Quantification of kinetics of Erk activation (pErk) normalized to total Erk expression. Two independently derived MEF cell lines for each genotype (E2 & E7 for WT and E3 & E5 for KO) were used in these experiments. Error bars represent standard deviation of pooled data from both sets of clones.
Ubiquitylation rNedd4-1
hNedd4-1
hNedd4-2
Supplementary Fig S1
Binding rNedd4-1
hNedd4-1
hNedd4-2
Supplementary Fig S2
A. Binding WT RxxQE-mut ____________ ___________
transfection __________ HA-LAPTM5(WT) HA-LAPTM5(YA) Flag-CACNB1 V5-hNedd4-2(WT) V5-hNedd4-2(CS)
+ +
+ +
-
- - + + - + - -
- - - - - - - - - - + ML + + + ML - ML - + - ML + - - - + -
WT RxxQE-mut ____________ ___________
transfection __________ HA-LAPTM5(WT) HA-LAPTM5(YA) Flag-ANXA9 T7-rNedd4-1(WT) T7-rNedd4-1(CS)
+ - + - - + +
- - - - - + + - - + - - -
- - + ML - ML + -
+ -
- - - - + + ML + - ML - + -
rNedd4-1 -> 13095-
130hNedd4-2 -> 95-
_______ ___________________________ IP: anti-HA anti-Flag _____________________________________ Blot: anti V5
_______ ___________________________ IP: anti-HA anti-Flag _____________________________________ Blot: anti -T7
B. Ubiquitylation transfection __________
transfection __________ Flag-CACNB1 V5-hNedd4-2(WT) V5-hNedd4-2(CS)
Flag-ANXA9
WT RxxQE-mut _______ ________ -
+ - +
-
T7-rNedd4-1(WT) T7-rNedd4-1(CS)
+ - +
170130IP: anti-Flag blot: anti-His (Ub) 9572-
170-
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-
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-
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55-
IP: anti-Flag blot: anti-Flag
IP: anti-Flag 130blot: anti-His (Ub)
WT RxxQE-mut _______ ________
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