Keywords

Phosphorylation, Serine/Threonine Kinase, DFG+1, Kinase Specificity, Catalysis

Reference

DOI: 10.1016/j.molcel.2013.11.013

Abstract

Eukaryotic protein kinases are often categorized as tyrosine or serine-threonine specific, but many serine-threonine kinases display strong preference for either Ser or Thr as phosphoacceptor residues. This paper identifies a single residue located in the kinase activation segment, immediately following the conserved DFG motif — termed the “DFG+1” residue — as a major determinant of this specificity.

Mutation of this DFG+1 residue is sufficient to switch phosphorylation site preference between Ser and Thr in multiple kinases (e.g., PAK4 and MST4). Structural studies show that phosphoacceptor residue preference is driven by conformational compatibility rather than binding strength, explaining how subtle differences between Ser and Thr determine kinase substrate selectivity.

Notes

1. Background and General Concept

  • Kinase substrate specificity is influenced by multiple mechanisms:
    • Colocalization, scaffold proteins, adaptor units, direct interactions.
  • Ser-Thr kinases (80% of human kinome) usually have conserved active site residues suited for these residues.
  • Some kinases prefer Ser (e.g., PKA), others prefer Thr (e.g., LKB1), and some show no preference.
  • The “DFG+1” residue in the activation segment is now identified as a key determinant for Ser/Thr preference.

2. Main Findings

  • The DFG+1 residue strongly influences kinase phosphoacceptor specificity.
  • Ser-specific kinases (e.g., PAK4) have large hydrophobic residues at DFG+1 (e.g., Phe, Leu, Met).
  • Thr-specific kinases (e.g., MST4) often have β-branched residues (e.g., Ile).
  • Non-selective kinases usually have smaller residues (e.g., Leu, Ser).

Experimental Highlights:

  • Mutating DFG+1 switches kinase preference:
    • PAK4 (Ser-specific) F461V mutation turns it into Thr-specific.
    • MST4 (Thr-specific) V165F mutation makes it Ser-specific.
  • Tested in multiple kinases: MST, PAK, Snf1, PKA — consistent shift in specificity.
  • Even predicts ROCK1’s Thr preference, an outlier in AGC family.

3. Structural and Biochemical Mechanism

  • Crystal structures of PAK4 WT and F461V mutant bound to Ser/Thr peptides reveal conformational basis:

    • WT PAK4’s DFG+1 Phe positioned close to Ser for optimal phosphate transfer.
    • Thr’s extra methyl group clashes, forcing Phe to rotate ∼75°, disrupting ATP positioning — explaining Ser preference.
    • In F461V mutant, steric clash is reduced, allowing better Thr phosphorylation.

  • Preference is based on conformational fit, not binding strength — catalytic alignment is key.

  • Kinetic data (kcat/Km) confirms DFG+1 swaps substrate preference in line with structural predictions.

4. Broader Implications

  • DFG+1 residue can predict Ser/Thr specificity across kinases — useful for functional classification.
  • Specificity for Ser or Thr matters in vivo — affects kinase-substrate pairing and signaling outcomes.
  • This finding helps understand kinase specificity “rules”, essential for interpreting signaling networks.
  • Relevant for designing selective kinase inhibitors and studying disease-linked kinase mutations.

5. Sth else

  • The kcat/Km ratio is used for evaluating catalytic efficiency — higher values mean more efficient enzymes.
  • Quote I like:

“The identity of the phosphoacceptor residue greatly affects the efficiency of substrate phosphorylation in living cells. Therefore, having a preferred phosphoacceptor residue is crucial for Ser-Thr kinases to target specific substrates. However, the exact principles governing this specificity is yet to be defined.”

  • Very beautiful paper : )

Take-home Messages

  • The DFG+1 residue is a central determinant of Ser/Thr kinase phosphoacceptor specificity.
  • Mutations at this site can switch preference between Ser and Thr.
  • Structural alignment for catalysis drives specificity, not just substrate binding affinity.
  • Rules of specificity can now be predicted from sequence, with implications for signaling and drug design.
  • The conformational basis of specificity emphasizes the role of subtle protein-ligand geometry in determining biological function.