N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

Abstract

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of protein kinases. The SRPKs (SR-specific protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine-proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1 (SR protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2-3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

Figure 1. CLK1 N-terminal sequences regulate RS domain binding & phosphorylation

A) CLK1 N-terminus. B) Phosphorylation of SRSF1 (0.15 μM) using ³²P-ATP (50 μM) and 50 nM CLK1 or CLK1(ΔN) is monitored by SDS-PAGE autoradiography. Time-dependent phosphorylation is fit with an amplitude and rate constant of 18 sites and 0.08 min⁻¹ for CLK1 (●) and 6 sites and 0.13 min⁻¹ for CLK1(ΔN) (○), respectively. C) Enzyme Doping. SRSF1 (60 nM) is phosphorylated using 30 nM CLK1 (●) and CLK1(ΔN) (○) and after 60 minutes, additional CLK1 (▲) or CLK1(ΔN) (△) are added to the CLK1(ΔN) reaction. D) The interaction of His-tagged CLK1 and CLK1(ΔN) with GST-SRSF1 is monitored on g-agarose beads using an anti-His antibody in pull-down assays. I = Input, PD = pull down. E) Competition Experiments. The phosphorylation of 50 nM SR(ΔRRM1) is monitored with varying amounts SRSF1 using CLK1 (●) and CLK1(ΔN) (○). The data are fit to equation (1) to obtain ^appK_I of 50 and 320 nM for CLK1 and CLK1(ΔN). F) Bar Graphs. Total number of phosphorylation sites and true binding affinities of SRSF1 are plotted for CLK1 and CLK1(ΔN). The error bars for the total number of sites were obtained from triplicate measurements.

Figure 2. Phosphorylation regiospecificity of the SRSF1 RS domain

A) Footprinting Strategy. LysC cleavage of SR(R224K) generates two major fragments corresponding to the N- and C-terminal halves of the RS domain. B) LysC cleavage. After complete phosphorylation with CLK1 and CLK1(ΔN) and ³²P-ATP, the N- and C-terminal halves of SR(R214K), generated by LysC treatment, are resolved by SDS-PAGE. The relative phosphoryl contents of the N- and C-terminal fragments [N/C] are calculated by a ratio of CPMs in the two bands. C) Phosphoryl contents of N & C are calculated using the total phosphoryl contents of uncleaved SR(R224K) and the N/C ratios. D) Ser-to-Ala Mutants in the SRSF1 RS Domain. E) Phosphorylation kinetics. The time-dependent data for CLK1 are fit to a single exponential function to obtain a rate constant and amplitude of 0.09 min⁻¹ and 12 sites for SR(4SA205) (●), 0.12 min⁻¹ and 10 sites for SR(4SA215) (○), 0.06 min⁻¹ and 9 sites for SR(4SA221) (▲), 0.08 min⁻¹ and 9 sites for SR(4SA221) (△), respectively. For CLK(ΔN), a rate constant and amplitude of 0.14 min⁻¹ and 5.1 sites for SR(4SA205), 0.09 ± 0.01 min⁻¹ and 6.3 sites for SR(4SA215), 0.06 min⁻¹ and 7 sites for SR(4SA221), 0.11 min⁻¹ and 7.2 sites for SR(4SA221), respectively, were obtained. The dashed lines in both plots were taken from Fig. 1B and represent SRSF1 phosphorylation. F) Bar graph showing total number of phosphates added in the presence of CLK1 and CLK(ΔN).

Figure 3. CLK1 N-Terminus interacts with the SRSF1 RS domain

A) GST-tagged Constructs of SRSF1. B) Pull-down assays using His-N and GST-tagged constructs on g-agarose resin. An asterisk denotes an impurity in the His-N preparation and a double asterisk denotes an impurity in the GST-SR(ΔRS) preparation. C) Pull-down assays using GST-N and His-tagged kinases and g-agarose resin. Kinases are probed using an anti-His antibody. I = Input, PD = pull down. Vertical dashed line indicates where lane from the same gel is spliced.

Figure 4. Phosphorylation and SRSF1 binding to CLK1

GST-SRSF1, bound to g-agarose resin, is used to pull down His-CLK1 (A), His-CLK1(ΔN) (B), and His-SRPK1 (C) in the presence of pre-phosphorylation with ATP. The His-tagged kinases were detected using an anti-His antibody. I = Input, PD = pull down. D) Autoradiogram showing phosphorylation of GST-SRSF1 by the kinases.

Figure 5. Phosphorylation-dependent binding of SRSF1 to the Ron ESE

A) CLK1 phosphorylation induces cooperative binding of SRSF1 to the Ron ESE. Fraction bound is plotted against the total concentration of unphosphorylated (●) and CLK1-phosphorylated SRSF1 (△). The values of N and K_0.5 are 1.2 ± 0.1 and 66 ± 2 nM for unphosphorylated SRSF1 and 2.0 ± 0.10 and 65 ± 2 nM for CLK1-phosphorylated SRSF1. B) CLK1(ΔN) phosphorylation does not induce cooperative binding of SRSF1 to the Ron ESE. Fraction bound is plotted against the total concentration of unphosphorylated (from panel A) and CLK1(ΔN)-phosphorylated SRSF1 (□). The values of N and K_0.5 are 1.2 ± 0.1 and 96 ± 5 nM for CLK1(ΔN)-phosphorylated SRSF1.

Figure 6. Effects of CLK1 N-Terminus on the phosphorylation of several SR proteins

A) RS domain sequences. B–D) CLK1 and CLK1(ΔN) are incubated with SRSF2 (B), SRSF5 (C), Tra2β1 (C) and ³²P-ATP and the reaction is monitored by SDS-PAGE autoradiography. The amount of ³²P incorporated in the presence of CLK1 (●) and CLK1(ΔN) (■) are plotted as a function of time. For SRSF2, amplitudes and rate constants of 0.58 μM and 0.050 min⁻¹ for CLK1 and 0.25 μM and 0.095 min⁻¹ for CLK1(ΔN). are obtained For SRSF5, amplitudes and rate constants of 0.45 μM and 0.038 min⁻¹ for CLK1 and 0.19 μM and 0.039 min⁻¹ for CLK1(ΔN) are obtained. For Tra2β1, amplitudes and rate constants of 0.65 μM and 0.081 min⁻¹ for CLK1 and 0.42 μM and 0.030 min⁻¹ for CLK1(ΔN) are obtained.

Figure 7. Model for induction of SR protein hyper-phosphorylated state by the CLK1 N-terminus

N-terminus of CLK1 can interact with RS domain in both unphosphorylated and phosphorylated states, thereby inducing the hyper-phosphorylated states of SR proteins. In the absence of the N-terminus [CLK1(ΔN)], only hypo-phosphorylated state is possible.