Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64

PLoS One. 2011;6(11):e28100. doi: 10.1371/journal.pone.0028100. Epub 2011 Nov 23.

Abstract

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / metabolism
  • Amino Acid Motifs
  • Animals
  • Catalytic Domain*
  • Cytoskeleton / metabolism
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / embryology
  • Drosophila melanogaster / enzymology*
  • Embryo, Nonmammalian / metabolism
  • Female
  • Fertility
  • Heterozygote
  • Mutation / genetics*
  • Mutation, Missense / genetics
  • Oocytes / cytology
  • Oocytes / metabolism
  • Protein-Tyrosine Kinases / chemistry*
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / chemistry*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Structure-Activity Relationship

Substances

  • Drosophila Proteins
  • Proto-Oncogene Proteins
  • Protein-Tyrosine Kinases
  • Src64B protein, Drosophila