Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity

Abstract

Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the "DFG+1" residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

Graphical abstract

Figure 1

The DFG+1 Residue Controls Ser-Thr Phosphosite Specificity For the kinases and peptide substrates indicated, the relative phosphorylation rates, as determined by radiolabel incorporation assay, are shown as a percentage of the maximally active substrate. The phosphorylation site residue is indicated by the color and location of the bar (Ser, green/left; Thr, red/right). (A) PAK4 and MST4 DFG+1 mutants exchange phosphorylation site specificity. Relative k_cat/K_m values for WT PAK4, PAK4^F461V, WT MST4, and MST4^V165F were derived from data shown in Table 1. (B) Relative reaction rates for phosphorylation of peptides with the sequence ALARAA(X)AAALAKKK at 40 μM, where X is Ser or Thr as indicated, by yeast Snf1 and the indicated mutants. Results are the mean ±SEM from three separate determinations. (C) Relative rates of WT PKA and PKA^F187V phosphorylation of 10 μM peptide (GGRRRRR(X)WYFGGGK). Results show mean ±SEM of triplicate samples from a representative experiment. (D) Relative rates of phosphorylation of the peptides ARKRERAY(X)FGHHA at 5 μM with the indicated phosphoacceptor residue by ROCK1. Results show mean ±SEM of duplicate samples from a representative experiment. Absolute reaction rates for (B)–(D) are provided in Table S3. See also Figure S1 and Tables S1 and S2.

Figure 2

The DFG+1 Residue Controls Protein Phosphorylation in Cells (A) GST-tagged BAD was transiently expressed in HEK293 cells with GFP-tagged WT PAK4 or PAK4^F461V catalytic domain. Following serum starvation and treatment with wortmannin to reduce phosphorylation by endogenous kinases, GST-BAD was purified from cell lysates and analyzed by immunoblotting using phosphospecific antibodies. Quantified signal intensities for BAD pSer112 normalized to the total BAD signal are shown below as the ratio to the background signal. (B) Phosphorylation of GST-tagged BAD^S112T following coexpression with WT PAK4 and PAK4^F461V was analyzed as in (A). A 5-fold greater quantity of BAD^S112T compared to WT BAD was loaded on the gel to compensate for the reduced reactivity of the BAD pSer112 antibody.

Figure 3

Structural Analysis of PAK4 and PAK4^f461v Complexes with PAKtide-S and PAKtide-T (A) Overall structure of PAK4 in complex with a PAKtide peptide. WT PAK4 with PAKtide-S is shown, with PAK4 in cartoon format and PAKtide-S in stick format. PAK4 is colored blue and PAKtide-S is colored green. DFG+1 residue Phe461 is indicated and shown in stick format. Phosphoacceptor residue Ser0 is indicated. Nucleotide is shown in stick format. Kinase N- and C-lobes are indicated. A box indicates the region shown in (B)–(E). (B–E) Close-ups of (B) PAK4 WT with PAKtide-S, (C) PAK4 WT with PAKtide-T, (D) PAK4^F461V with PAKtide-T, and (E) PAK4^F461V with PAKtide-S are shown. The phosphoacceptor residue Ser0 or Thr0 and the DFG+1 residue Phe461 or Val461 are shown as spheres. Structural figures were generated using CCP4mg (McNicholas et al., 2011). See also Figure S3.