Figure 2. ARIH1 is capable of generating modest poly-ubiquitin chains onto SCF-bound substrate at either saturating or more physiological concentrations.
(a) Typical conditions for the single-encounter quench flow ubiquitylation reactions used to estimate the rates of ubiquitin transfer. (b) Autoradiogram of a Cyclin E peptide ubiquitylation reaction with ARIH1 levels (2.5 μM) sufficient to saturate the SCF complex, where S0 represents unmodified substrate, S1 represents substrate modified with one ubiquitin, etc. Each time-point was performed in duplicate technical replicates. (c) Data points and fit to the kinetic model of the reaction in (b) for substrate (S0) and two products (S1 and S2). (d) Same as (b), except more physiological ARIH1 levels (Table 2) were used in the assay. (e) Same as (b), except with β-Catenin peptide substrate and SCFβTRCP. (f) Same as (e), except with more physiological ARIH1 levels. Figure 2—figure supplements 1–9 show the reactions and/or the fit of the data to the model for UBE2D3, UBE2R2, and combinations with ARIH1 or UBE2D3. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—source data 1. - replicate data for the graphs shown in Figure 2 and Figure 2—figure supplements 1–9.
elife-51163-fig2-data1.xlsx (532KB, xlsx)
Figure 2—figure supplement 1. Estimation of the rates of ARIH1-catalyzed ubiquitin transfer to SCF-bound substrate.
(a) Data points and fit to the kinetic model of a ubiquitylation reaction containing Cyclin E peptide and physiological ARIH1 levels (Table 2) (see Figure 2 - panel d for the autoradiogram). Duplicate data points are shown from technical replicates. (b) Same as (a), except with β-Catenin peptide substrate levels that are saturating for SCFβTRCP (autoradiogram shown in Figure 2 - panel e). (c) Same as (b), except with more physiological ARIH1 levels (also see Figure 2 - panel f).
Figure 2—figure supplement 2. UBE2D3 is capable of generating modest poly-ubiquitin chains onto SCF-bound Cyclin E substrate at either saturating or more physiological concentrations.
(a) Autoradiogram of a Cyclin E ubiquitylation reaction with UBE2D3 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model of the reaction in (a) for substrate (S0) and mono-ubiquitylated product (S1). Duplicate data points from technical replicates are shown. (c,d) Same as (a,b), except with more physiological UBE2D3 levels. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 3. UBE2D3 is capable of generating modest poly-ubiquitin chains onto SCF-bound β-Catenin substrate at either saturating or more physiological concentrations.
(a) Autoradiogram of a β-Catenin peptide ubiquitylation reaction with UBE2D3 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model for the reaction in panel a. Duplicate data points from technical replicates are shown. (c,d) Same as (a,b), except with more physiological UBE2D3 levels. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 4. UBE2R2 generates robust poly-ubiquitin chains onto SCF-bound substrates when its levels are sufficient to saturate SCF.
(a) Autoradiogram of a Cyclin E ubiquitylation reaction with saturating UBE2R2 levels. Notice that mono-ubiquitylated substrate (S1) is barely detectible since it is very rapidly converted to S2. (b) Data points and fit to the kinetic model for the reaction in panel a. Estimation of the rate of ubiquitin transfer from UBE2R2 ~ ubiquitin to S1 was done independently, since S1 levels are only approximately 1% of the total signal and could not be reliably fit to the model. Duplicate data points from technical replicates are shown. (c) Autoradiogram of a mono-ubiquitylated Cyclin E peptide (S1) ubiquitylation reaction containing SCFFBW7 and saturating levels of UBE2R2. Reactions were also carried out in the presence of lysine-less (K0) ubiquitin, resulting in a single product (S2). (d) Data points and fit to the single, exponential decay of S1 and the formation of S2 from the reaction in panel c. Duplicate data points from technical replicates are shown. (e, f) Same as in (a, b), except with β-Catenin peptide substrate and SCFβTRCP. (g, h) Same as in (c, d), except with mono-ubiquitylated β-Catenin peptide substrate and SCFβTRCP. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 5. UBE2R2 activity is negligible when assayed at physiological conditions.
(a) Autoradiogram of a Cyclin E ubiquitylation reaction with more physiological UBE2R2 levels. (b) Same as in (a), except with β-Catenin peptide substrate and SCFβTRCP. The data are representative of duplicate technical replicates. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 6. The combination of ARIH1 with UBE2R2 protein results in Cyclin E substrates modified with longer poly-ubiquitin chains than with ARIH1 alone.
(a) Autoradiogram of a Cyclin E ubiquitylation reaction with ARIH1 and UBE2R2 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model for the reaction shown in panel a. Duplicate data points from technical replicates are shown. (c, d) Same as (a, b), except with more physiological ARIH1 and UBE2R2 levels. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 7. The combination of ARIH1 with UBE2R2 protein results in β-Catenin substrates modified with longer poly-ubiquitin chains than with ARIH1 alone.
(a) Autoradiogram of a β-Catenin peptide ubiquitylation reaction with ARIH1 and UBE2R2 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model for the reaction in panel a. Duplicate data points from technical replicates are shown. (c, d) Same as (a, b), except with more physiological ARIH1 and UBE2R2 levels. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 8. The combination of UBE2D3 with UBE2R2 protein results in substrates modified with longer poly-ubiquitin chains than with UBE2D3 alone.
(a) Autoradiogram of a Cyclin E ubiquitylation reaction with UBE2D3 and UBE2R2 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model for the reaction in panel a. Duplicate data points from technical replicates are shown. (c, d) Same as (a, b) except with more physiological UBE2D3 and UBE2R2 levels. The enzyme concentrations have been provided in Supplementary file 2.
Figure 2—figure supplement 9. The combination of UBE2D3 with UBE2R2 protein results in substrates modified with longer poly-ubiquitin chains than with UBE2D3 alone, especially in the presence of β-Catenin peptide substrate.
(a) Autoradiogram of a β-Catenin peptide ubiquitylation reaction with UBE2D3 and UBE2R2 levels sufficient to saturate the SCF complex. (b) Data points and fit to the kinetic model for the reaction in panel a. Duplicate data points from technical replicates are shown. (c, d) Same as (a, b), except with more physiological UBE2D3 and UBE2R2 levels. The enzyme concentrations have been provided in Supplementary file 2.