Fig. 4: Investigation of FIKK1, FIKK4.1 and FIKK4.2 substrate specificities.

a, Left: a representation of FIKK phosphoproteome peptides membrane, each dot represents one target-peptide species. Yellow, FIKK1 targets; orange, FIKK4.1 targets; green, FIKK4.2 targets; blue, FIKK10.2 targets; red, host cell peptides phosphorylated in iRBCs. The list of peptides with sequences is provided in Supplementary Table 10. Right: FIKK1 activity on the phosphoproteome peptides membrane. b, Left: correlation of FIKK1 activity on the phosphoproteome peptides membrane (log₁₀ transformed) against FIKK1 motif score (matrix similarity score) for each peptide (n = 163). Pearson’s correlation for the y = log(x) curve. Right: FIKK1 phosphorylation signal (Phosphosignal) (log₁₀ transformed) for peptides with or without matching FIKK1 motif, for peptides with an S (n = 269, Cohen’s D = 1.2, P = 1.5 × 10⁻⁸, Wilcoxon test, one sided) or T phosphoacceptor (n = 95, Cohen’s D = 0.57, P = 0.13, Wilcoxon test, one sided). The centre line shows the median, the box limits the upper and lower quartiles, the whiskers show 1.5× the interquartile range and each point indicates an outlier. c, Left: FIKK1 specificity logo derived from the OPAL membrane. Right: FIKK1 specificity logo derived from natural peptides phosphorylated by FIKK1 on the peptide membrane. d, Left: FIKK4.1 activity (log₁₀ transformed) against predicted target peptides of FIKK4.1 (orange) and of FIKK4.2 (green) (n = 174 peptides, Cohen’s D = 0.57, P = 4.0 × 10⁻³, Wilcoxon test, one sided). Right: FIKK4.2 activity (log₁₀ transformed) against predicted target peptides of FIKK4.1 (orange) and of FIKK4.2 (green) (n = 174 peptides, Cohen’s D = 0.10, P = 0.77, Wilcoxon test, one sided). The black dot shows the median and the black line shows the upper and lower quartiles. e, FIKK4.1 and FIKK4.2 activity on KAHRP_345, PTP4_1091 and PIESP2_267. The results are represented as mean ± s.e.m. fold change compared with the no substrate luminescent signal. Statistical significance was determined using a one-way analysis of variance followed by Dunnett’s multiple comparison post-test (for FIKK4.1, KAHRP_345 versus no substrate, P < 0.0001; PTP4_1091 versus no substrate, P < 0.0001; PIESP2_267 versus no substrate, P = 0.0001; FIKK4.2, KAHRP_345 versus no substrate, P = 0.9658; PTP4_1091 versus no substrate, P = 0.9069; PIESP2_267 versus no substrate, P = 0.4315) (n = 3 biological replicates). f, FIKK4.1 and FIKK4.2 PWM logos made using data from Supplementary Fig. 3. Values are log₂ transformed. A positive value depicts favoured amino acids and a negative value depicts disfavoured amino acids. See Fig. 3 caption for the colour code and Supplementary Fig. 8 for log₂-transformed PWM logos for all recombinant FIKK tested.