Recruiting a silent partner for activation of the protein kinase SRPK1

Abstract

The SRPK family of protein kinases regulates mRNA splicing by phosphorylating an essential group of factors known as SR proteins, so named for a C-terminal domain enriched in arginine-serine dipeptide repeats (RS domains). SRPKs phosphorylate RS domains at numerous sites altering SR protein subcellular localization and splicing function. The RS domains in these splicing factors differ considerably in overall length and dipeptide layout. Despite their importance, little is known about how these diverse RS domains interact with SRPKs and regulate SR protein phosphorylation. We now show that sequences distal to the SRPK1 consensus region in the RS domain of the prototype SR protein SRSF1 are not passive as originally thought but rather play active roles in accelerating phosphorylation rates. Located in the C-terminal end of the RS domain, this nonconsensus region up-regulates rate-limiting ADP release through the nucleotide release factor, a structural module in SRPK1 composed of two noncontiguous sequence elements outside the kinase core domain. The data show that the RS domain in SRSF1 is multifunctional and that sequences once thought to be catalytically silent can be recruited to enhance the efficiency of SR protein phosphorylation.

Figure 1

C-terminal residues in the RS domain increase SRSF1 turnover. (A) Wild-type and mutant forms of the SR protein SRSF1. Residues 204–248 of the RS domain are shown, and the RRMs (RRM1 and RRM2) are omitted. (B) Structure of SRPK1 in complex with SRSF1. The two lobes of the kinase domain [subdomains I–VI (N-lobe) and VII–XI (C-lobe)] are shown in gray. SRPK1 lacks its N-terminus (dotted red line) and most of the SID with the exception of helix Sα1 (red). SRSF1 was crystallized without RRM1 and residues 220–248 (RS2). Disordered residues 211–219 (part of RS1) and 197–200 (linker between RRM2 and RS domain) are shown as dotted green lines. Residues 201–210 (N-terminus of RS1) are visible in the docking groove in the C-lobe of the kinase domain. (C) Plot of initial velocity versus SRSF1 (●), SR(1–226) (○), SR(ΔRS2) (▲), SR(RA1) (△), SR(RA2) (■), SR(RA12) (□). Data fits are included in Table 1. (D) Bar graph showing turnover numbers (k_cat) for each substrate.

Scheme 1

Figure 2

RS2 controls the release rate for ADP. (A) Glycerol effects on SR(ΔRS2) phosphorylation. Plots of initial velocity versus ATP at 0% (●), 10% (○), 25% (▲), and 30% (△) sucrose. (B) Relative k_cat and k_cat/K_ATP (ratios in the absence and presence of sucrose) versus relative solvent viscosity for the data in panel A. Slopes of 1.0 and 1.2 are obtained for relative k_cat and k_cat/K_ATP. Dotted lines represent theoretical slope values of 0 and 1. (C) Pre-steady-state kinetic transients for SR(ΔRS2) (●) and SRSF1 (▲) phosphorylation. SRPK1 (0.25 μM) is mixed with SR protein (0.5 μM) and ATP (600 μM) in the rapid quench flow machine. The data are fit to eq 1 to obtain values of 0.23 ± 0.02 μM, 12 ± 3 s^–1, and 0.28 ± 0.01 s^–1 for α, k_b, and k_L, respectively, for SR(ΔRS2) and values of 0.22 ± 0.03 μM, 19 ± 8 s^–1, and 1.0 ± 0.01 s^–1 for α, k_b, and k_L, respectively, for SRSF1. (D) C_ATT_RAP experiment. SRPK1 is preincubated with SR(ΔRS2) in the absence (●) and presence (▲) of 120 μM ADP in one syringe (60 μM in reaction) of the rapid quench machine and then mixed with ATP (600 μM in reaction) to start the reaction. The data in panel D are simulated using DynaFit and the mechanism in Scheme 2 to obtain values of 0.3 s^–1, 18 s^–1, and 0.3 s^–1 for k_off, k₂, and k₃, respectively (solid lines). A value of 19 mM^–1 s^–1 for the k₁ was used for both simulations. Additional simulations in the presence of ADP are displayed in which k_off is increased to values of 0.37 s^–1 (···), 0.45 s^–1 (−–−), and 0.6 s^–1 (— - —).

Scheme 2

Figure 3

RS2 activates SRPK1 by binding outside the active site. (A) SRSF1 constructs. (B) SR(RS2) is a substrate for SRPK1. Initial velocity data for SR(RS2) phosphorylation are collected using 5 μM ATP and fit to K_SR and k_cat values of 340 ± 100 nM and 0.9 ± 0.1 min^–1. (C) Competition with SR(RS2). The relative initial velocities for the phosphorylation of 50 nM SRSF1 (▲) or SR(ΔRS2) (●) are monitored using 5 μM ATP and varying amounts of SR(RS2) (0–2000 nM). For SRSF1, the data are fit to eq 2 to obtain a K_I of 200 ± 20 nM for SR(RS2) using a fixed value for K_SR of 110 nM (Table 1). For SR(ΔRS2) phosphorylation, the data are fit to eq 3 to obtain K_A of 330 ± 70 nM, K_I of 320 ± 90 nM, and γ of 5 ± 0.8 for SR(RS2) using a fixed value for K_SR (25 nM). (D) Initial velocity kinetics for SR(ΔRS2) phosphorylation in the absence (▲) and presence (●) of 200 nM SR(RS2). Values for k_cat and K_SR are 0.33 ± 0.05 s^–1 and 30 ± 9 nM in the absence and 1.3 ± 0.21 s^–1 and 28 ± 11 nM in the presence of SR(RS2) and 100 μM ATP.

Scheme 3

Figure 4

Nucleotide release sequences regulate RS2-dependent activation of SRPK1. (A) SRPK1 deletion constructs. Helix Sα1 in the SID is shown in striped red. (B) SR(RS2) is a substrate for SRPK1(ΔN) (▲) and SRPK1(ΔS) (●). Initial velocity data are collected using 5 μM ATP and fit to a k_cat/K_SR value of 0.66 ± 0.05 μM^–1 min^–1 for SRPK1(ΔS) and 0.25 ± 0.07 μM^–1min^–1 for SRPK1(ΔN). (C) Competition data. The relative initial velocities for SR(ΔRS2) phosphorylation using SRPK1(ΔS) (▲) and SRPK1(ΔN) (●) are plotted as a function of SR(RS2). The data for SRPK1(ΔS) were fit to eq 2 using a K_SR of 110 nM to obtain a K_I of 1300 ± 100 nM. (D–F) Plots of initial velocity for SR(ΔRS2) phosphorylation using SRPK1(ΔN) (▲,△), SRPK1(ΔS) (●,○), SRPK1(ΔS_INT) (■,□), and SRPK1(ΔSα1) (▼,▽) in the absence (filled symbols) and presence (open symbols) of 200 nM SR(RS2). The kinetic parameters are displayed in Table 3

Figure 5

Model for RS2-dependent activation of SRPK1. The SRPK1–SRSF1 complex undergoes fast phosphoryl transfer followed by rate-limiting ADP dissociation (nucleotide release). Translocation reflects movement of the RS domain in the docking groove and active site and ATP binding for the delivery of subsequent phosphates. A 10-fold enhancement in net SR protein phosphorylation is achieved in a two-step process where step 1 involves a 3-fold increase resulting from the effect of the NRF on the nucleotide pocket and step 2 involves an additional 3-fold increase in ADP release resulting from NRF–RS2 modulatory interactions.