Applying the brakes to multisite SR protein phosphorylation: substrate-induced effects on the splicing kinase SRPK1

Abstract

To investigate how a protein kinase interacts with its protein substrate during extended, multisite phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates ~10 serines in the arginine--serine-rich domain (RS domain) of the SR protein SRSF1 in a C- to N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t(1/2) = 0.1 s) followed by slower, multisite phosphorylation at the remaining serines (t(1/2) = 15 s). Mutagenesis experiments suggest that efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic network between the RS domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multisite phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate, and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension, and termination events associated with prolonged, multisite phosphorylation.

Figure 1. Structure and Phosphorylation Mechanism of SRSF1

A) SRSF1 Domain Structure. SRSF1 has two RRMs and a C-terminal RS domain composed of RS1 and RS2 segments. B) Structure of SRPK1 core with AMPPNP and the N-terminal portion of RS1 segment (R₂₀₄SRSRSR) bound to the docking groove in the large lobe. Arginines are colored blue and serines are colored red. A hydrogen bond is formed between the hydroxyl of Ser207 and the backbone carbonyl of Ser209. RRM2 is not displayed in this depiction. C) Phosphorylation Model. SRPK1 binds SRSF1 with high affinity using an electronegative docking groove that initially recognizes the N-terminal portion of RS1. Upon progressive phosphorylation, RS1 is threaded through the docking groove and into the active site in a C-to-N-terminal direction. In later phosphorylation stages, β4 from RRM2 unfolds and binds in the docking groove and phosphoserines in RS1 are stabilized by an electropositive site (P+2 site)

Figure 2. Phosphorylation of Full-Length and Truncated SRSF1 Using Rapid Quench Flow Methods

A&C) Single Turnover Experiments. A complex of SRPK1 (2 μM) and SRSF1 (0.2 μM) (A) or RS domain (0.4 μM) (C) in one syringe is mixed with ATP (200 μM) in the second syringe in the rapid quench flow machine. Final concentrations are 1 μM SRPK1, 100 μM ATP, 0.1 μM SRSF1 in (A) or 0.2 μM RS domain in (C). For SRSF1, the data are fit to values of 6 ± 2 sec⁻¹ for k_p, 0.042 ± 0.010 sec⁻¹ for k_mp, 0.10 ± 0.03 μM for α_p and 0.80 ± 0.10 μM for α_mp. For the RS domain, the data are fit to values of 7 ± 3 sec⁻¹ for k_p, 0.048 ± 0.010 sec⁻¹ for k_mp, 0.20 ± 0.04 μM for α_p and 1.8 ± 0.10 μM for α_mp. B&D) Pre-Steady-State Kinetic Experiments. A complex of SRPK1 (0.25 or 0.5 μM) and SRSF1 (1 μM) in (B) or SRPK1 (0.5 μM) and RS domain (5μM) in (D) is mixed with ATP (200 μM) in the rapid quench flow machine. Final concentrations in (B) are 0.125 (●) or 0.25 μM (○) SRPK1, 0.5 μM SRSF1 and 100 μM ATP. The data for SRSF1 phosphorylation at 0.125 and 0.25 μM SRPK1 are fit to equation (1) to obtain the respective values of 8 ± 2 and 11 ± 5 sec⁻¹ for k_b, 0.79 ± 0.11 and 0.82 ± 0.13 for α, and 0.85 ± 0.10, and 0.82 ± 0.13 sec⁻¹ for k_L. Final concentrations in (D) are 2.5 μM RS domain, 0.25 μM SRPK1 and 100 μM ATP. The data for RS domain phosphorylation are fit to equation (1) to obtain the respective values of 12 ± 3 sec⁻¹ for k_b, 0.80 ± 0.10 for a, and 0.75 ± 0.08 for k_L.

Figure 3. Enzyme-Substrate Interactions Support Phosphorylation Rates

A) Docking Groove. The phosphorylation of SRSF1 by a form of SRPK1 containing 6 mutations in the docking groove [SRPK1(6M)] was studied. The data for wild-type SRPK1 (●) are fit to values of 5.7 sec⁻¹ for k_p, 0.063 for k_mp, 1 site for α_p, and 9 sites for α_mp. For SRPK1(6M) (○) the data are fit to single exponential function with values of 0.023 sec⁻¹ for k_mp and 6.1 sites for α_mp. B) RS Domain. The phosphorylation of multiple Arg-to-Ala and Ser-to-Ala mutants in the RS domain of SRSF1 by SRPK1 were studied. For wild-type SRSF1 (●), values of 5 sec⁻¹ for k_p, 0.050 sec⁻¹ for k_mp, 1 site for α_p and 8.7 sites for α_mp are obtained. The data for the mutants are fit to single exponential functions with values of 0.0057 sec⁻¹ (k_mp) and 6.9 sites (α_mp) for SR(5RA222) (○), 0.018 sec⁻¹ (k_mp) and 6.0 sites (α_mp) for SR(4SA221) (▲), 0.023 sec⁻¹ (k_mp) and 6.0 sites (α_mp) for SR(4SA219) (△), 0.013 sec⁻¹ (k_mp) and 5.2 sites (α_mp) for SR(4SA215) (■), 0.009 sec⁻¹ (k_mp) and 5.3 sites (α_mp) for SR(4SA205) (□). C) Multi-Site Phosphorylation. The bar graph displays the ratio of k_mp and v_mp (k_mp x α_mp) for the mutants compared to SRSF1.

Figure 4. Positional Analyses of the Phosphoryl Transfer Rate

A) Pre-phosphorylation of SRSF1. Complexes of SRPK1 (1 μM) and SRSF1 (100 nM) are incubated with limiting ³²P-ATP (0-5 μM) for 20 min to attain 0, 1.2, 4, 6.3 and 8.3 phosphates (0P, 1P, 4P, 6P, 8P) onto the RS1 segment. B) Single Turnover Kinetics. Excess ³²P-ATP (100 μM) is added to the complexes generated in (A) and the phosphorylation of the remaining sites is monitored. The data are fit to values of 3.6 ± 1.0 sec⁻¹ for k_p, 0.050 ± 0.006 sec⁻¹ for k_mp, 1.0 ± 0.18 sites for α_p and 9.1 ± 0.35 sites for α_mp for 0P (●), 3.8 ± 1.2 sec⁻¹ for k_p, 0.050 ± 0.004 sec⁻¹ for k_mp, 1.1 ± 0.20 sites for α_p and 7.5 ± 0.25 sites for α_mp for 1P (○), 4.3 ± 1.5 sec for k_p, 0.040 ± 0.004 sec⁻¹ for k_mp, 1.1 ± 0.14 sites for α_p and 4.9 ± 0.35 sites for α_mp for 4P (▲), 3.0 ± 1.0 sec⁻¹ for k_p, 0.035 ± 0.004 sec⁻¹ for k_mp, 0.80 ± 0.05 sites for α_p and 2.9 ± 0.08 sites for α_mp for 6P (△) and 3.0 ± 1.1 sec⁻¹ for k_p, 0.040 ± 0.003 sec⁻¹ for k_mp, 0.32 ± 0.05 sites for α_p and 1.4 ± 0.25 sites for α mp for 8P (◆). C) Amplitude and Linear Rates. The values of α_p and v_mp (k_mp x α_mp) from (B) are plotted as a function of pre-phosphorylation state of SRSF1. D) SRPK1 & ATP Dependence. The 8P complex is generated as described in panel (A) and the reaction is then started with either 400 μM ³²P-ATP (●) or 4 μM SRPK1 (○). The data are fit to values of 4.0 ± 0.80 sec⁻¹ (k_p), 0.030 ± 0.004 sec⁻¹ (k_mp), 0.33 ± 0.06 sites (α_p) and 1.7 ± 0.06 sites (α_mp) at 400 μM P-ATP and 4.6 ± 0.82 sec⁻¹ (k_p), 0.031 ± 0.004 sec⁻¹ (k_mp), 0.67 ± 0.037 sites (α_p) and 1.3 ± 0.06 sites (α_mp) at 4 μM SRPK1. The 0P complex is also reacted with 600 μM ³²P-ATP (▲) and fit to values of 25 ± 8 sec⁻¹ (k_p), 0.043 ± 0.006 sec⁻¹ (k_mp), 0.91 ± 0.16 sites (α_p) and 9.0 ± 0.39 sites (α_mp)

Figure 5. Effects of Sucrose on the Steady-State Kinetic Parameters for SRPK1

Initial velocities for SRPK1 are plotted as a function of total SRSF1 at fixed ATP (A) or as a function of ATP at fixed SRSF1 (C) using 0% (●), 10% (○), 25% (▲), and 30% (△) sucrose. In the absence of sucrose, the k_cat and K_m for SRSF1 are 56 ± 5 min⁻¹ and 70 ± 6 nM using fixed ATP (100 μM) and the k_cat and K_m for ATP are 50 ± 5 min⁻¹ and 12 ± 2 μM using fixed SRSF1 (1 μM). The effects of added sucrose on the relative steady-state kinetic parameters [${\text{k}}_{\text{cat}}^{°}$/k_cat and (k_cat/K_m)°/(k_cat/K_m)] are plotted as a function of relative solvent viscosity (η^rel) under conditions of varying SRSF1 (B) or ATP (D). For varying SRSF1, ${\text{k}}_{\text{cat}}^{\eta }$ and (k_cat/K_m)^η are 1.2 ± 0.16 and 0.90 ± 0.10. For varying ATP, ${\text{k}}_{\text{cat}}^{\eta }$ and (k_cat/K_m)^η are 1.2 ± 0.18 and 1.0 ± 0.05. Initial velocities for SRPK1 as a function of Block Mutant are plotted in panel (E) at fixed ATP (100 μM). In the absence of sucrose, the k_cat and K_m for SRSF1 are 2.4 ± 0.25 min⁻¹ and 2.1 ± 0.60 μM. For varying Block Mutant,${\text{k}}_{\text{cat}}^{\eta }$ and (k_cat/K_m)^η are −.020 ± 0.043 and 0.090 ± 0.010.

Figure 6. Nucleotide Exchange Kinetics in the SRPK1-SRSF1 Complex

A) ATP-Dependent Kinetic Transients. A complex of SRPK1 (0.5 μM) and SRSF1 (1 μM) in one syringe is mixed with ATP (100 or 1000 μM) in the second syringe in the rapid quench flow machine. Final concentrations are 50 (●) or 500 μM (○) ATP, 0.25 μM SRPK1 and 0.5 μM for SRSF1. The data are simulated using DynaFit (26) and Scheme 2 to obtain values of 0.09 μM⁻¹sec⁻¹, 30 sec⁻¹, and 0.8 sec⁻¹ for k₁, k₂, and k₃, respectively. B) ADP Binding Affinity. Initial reaction velocities are measured as a function of ATP (6-800 μM) at fixed SRSF1 (1 μM) in the absence (●) and presence of 30 (○), 60 (▲), and 120 μM (△) ADP. K_I = 10 μM for ADP. C) ADP Catalytic Trapping From 0P. A complex of SRPK1 (0.5 μM) and SRSF1 (1 μM) with (○) or without (●) ADP (120 μM) in one syringe is mixed with ATP (1200 μM) in the second syringe of the rapid quench flow machine. Final concentrations are 600 μM ATP, 0.25 μM SRPK1, 0.5 μM SRSF1 and 60 μM ADP. The data in the absence of ADP are simulated using DynaFit and Scheme 2 to obtain values of 0.09 μM⁻¹sec⁻¹, 30 sec⁻¹, and 1.1 sec⁻¹ for k₁, k₂, and k₃, respectively. These values are then used to simulate the data in the presence of ADP using the mechanism in Scheme 3 and DynaFit to obtain a k_off for ADP of 1.1 sec⁻¹. D) ADP Catalytic Trapping from 4P. A complex of SRPK1 (2 μM) and SRSF1 (0.26 μM) is prequibrated for 20 min with 3 μM ³²P-ATP to generate a 4P complex. This complex is incubated with (○) or without (●) ADP (120 μM) in one syringe and then mixed with ATP (1200 μM) in the second syringe of the rapid quench flow machine. Final concentrations are 600 μM ATP, 1 μM SRPK1, 0.13 μM SRSF1 and 60 μM ADP. The data in the absence of ADP are simulated using DynaFit and Scheme 2 to obtain values of 0.04 μM⁻¹sec⁻¹, 2.7 sec⁻¹, and 0.10 sec⁻¹ for k₁, k₂, and k₃, respectively. These values are then used to simulate the data in the presence of ADP using the mechanism in Scheme 3 and DynaFit to obtain a k_off for ADP of 0.13 sec⁻¹.

Figure 7. Effects of Phosphorylation Progress on Individual Steps

A) Simulation of Progress Curves. The progress curves for 0P (○) and 4P (△) (data from Fig.4B) were simulated using DynaFit and a sequential mechanism in which 0P converts step-wise to 10P. The irreversible, net rate constants for each individual step are defined by reactant phosphorylation state (k_0P, k_1P, k_2P,…k_9P). Simulations for 0P include k_0P through k_9P whereas those for 4P include k_4P through k_9P. Values of 5, 1, 0.6, 0.45, 0.2, 0.15, 0.12, 0.1, 0.07, and 0.06 sec⁻¹ for k_0P, k_1P, k_2P, k_3P, k_4P, k_5P, k_6P, k_7P, k_8P, and k_9P, respectively, were used to simulate 0P. Values of 3, 0.5, 0.1, 0.06, 0.04, and 0.02 sec⁻¹ for k_4P, k_5P, k_6P, k_7P, k_8P, and k_9P, respectively, were used to simulate 4P. For comparison, the analytical fits to both curves are shown as dotted lines and their parameter fits are included in the legend of Fig. 4B. B) Change in Free Energy Reaction Coordinate Diagram As A Function of Phosphorylation. SRPK1 has high affinity for SRSF1, rapidly phosphorylates the first serine in the RS domain and slowly releases ADP (solid line). After several rounds of phosphorylation (6-8P), the affinity of the substrate declines significantly and the phosphoryl transfer and ADP release rates decrease (dashed line).

Scheme 1

Scheme 2

Scheme 3