Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C.
Activation of phosphatidylcholine-specific phospholipase D (PLD) constitutes an important part of the cellular response to agonist signaling. PLD1 is stimulated in vitro in a direct and synergistic manner by protein kinase C (PKC), ADP-ribosylation factor (ARF) and Rho family members. However, the direct and specific role of each of these effectors in agonist-stimulated PLD activation is poorly understood. We have used transposon mutagenesis to generate a library of PLD1 alleles containing random pentapeptide insertions. Forty-five alleles were characterized to identify functionally important regions. Use of an allele unresponsive to PKC, but otherwise seemingly normal, to examine coupling of PLD1 to a subset of G-protein-coupled receptors demonstrates for the first time direct stimulation of PLD1 in vivo by PKC and reveals that this direct stimulation is unexpectedly critical for PLD1 activation.
Zhang
Y
Y
Molecular and Cellular Biology Program, SUNY at Stony Brook, Stony Brook, NY 11794-8651, USA.
Altshuller
Y M
YM
Hammond
S M
SM
Hayes
F
F
Morris
A J
AJ
Frohman
M A
MA
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D011493:Protein Kinase C; D010739:Phospholipase D; C106801:phospholipase D1; D019204:GTP-Binding Proteins