Fig. 6.

Effects of proline mutations on SRSF1 binding and phosphorylation. (a) Mutations in RS domain prolines. Residues phosphorylated by SRPK1 are highlighted with brackets. (b) Single turnover kinetics. CLK1 (0.5 μM) and 100 μM ³²P-ATP are added to 0.2 μM SRSF1 (●), SR(P200A) (○), SR(P228A) (▲), SR(P235A) (△), SR(P239A) (■), SR(P228,235A) (□), SR(P200,228,235A) (▼) and SR(P235,239A) (▽). The data are fit to a single exponential to obtain rate constants and amplitudes of 0.60 ± .08 min⁻¹ and 18 ± 0.6 for SRSF1, 0.44 ± .05 min⁻¹ and 16 ± 0.6 for SR(P200A), 0.34 ± .06 min⁻¹ and 15 ± 0.6 for SR(P228A), 0.58 ± .04 min⁻¹ and 19 ± 0.4 for SR(P235A), 0.41 ± .02 min⁻¹ and 20 ± 0.4 for SR(P228,235A), 0.25 ± .02 min⁻¹ and 13 ± 0.39 for SR(P200,228,235A) and 0.26 ± .02 min⁻¹ and 17 ± 0.34 for SR(P235,239A), respectively. SR(P239A) was fit to a double exponential with amplitudes of 5 ± 0.4 and 15 ± 0.4 sites and rate constants of 1.1 ± .25 and 0.034 ± 0.002 min⁻¹, respectively. (c) Bar graph showing phosphoryl contents of mutants. Data are taken from the total amplitudes in (b). (d and e) Competition experiments for mutants using SRPK1 (d) and CLK1 (e). Fixed amounts of SR(ΔRRM1) (50 nM) are mixed with either CLK1 (10 nM) or SRPK1 (1 nM) and varying amounts of mutant SR proteins. Mutants are labeled as in (b), and the ^appK_I and K_I values are displayed in Table 1. (f) Bar graph showing K_I values for the mutants relative to SRSF1 for CLK1 and SRPK1.