Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects.
PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree of phenotypic variation with viability of mutant adult flies ranging from 0 to 96%, and ISNb motor nerve defects ranging from 11 to 57% [Desai and Purdy, 2003]. To determine whether mutations in Ptp69D affecting axon guidance and viability demonstrate losses of phosphatase activity and whether differences in catalytic potential underlie phenotypic variability, we expressed full-length wild-type and mutant PTP69D protein in Schneider 2 cells, and assessed phosphatase activity using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP). Detailed biochemical characterization of wild-type PTP69D, including an examination of sensitivity to various inhibitors, in vitro catalytic efficiency, and the pH-k(cat) profile of the enzyme, suggests a common tyrosine phosphatase reaction mechanism despite lack of sequence conservation in the WPD loop. Analysis of mutant proteins revealed that every mutant had less than 1% activity relative to the wild-type enzyme, and these rates did not differ significantly from one another. These results indicate that mutations in Ptp69D resulting in axon guidance defects and lethality significantly compromise catalytic activity, yet the range of biological activity exhibited by Ptp69D mutants cannot be explained by differences in catalytic activity, as gauged by their ability to hydrolyze the substrate DiFMUP.
Marlo
Joy E
JE
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. joy.marlo@vanderbilt.edu
Desai
Chand J
CJ
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000201:enzymology*; D001665:Binding Sites; D002460:Cell Line; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D054557:Receptor-Like Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011956:Receptors, Cell Surface / Q000378:metabolism; D013696:Temperature
D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D017493:Leukocyte Common Antigens; C482451:PTP69D protein, Drosophila; D017027:Protein Tyrosine Phosphatases; D054557:Receptor-Like Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2