Ca2(+)-enhanced phosphorylation of a chimeric protein kinase involved with bacterial signal transduction.
The Tar-EnvZ hybrid molecule (Taz1) is an inner membrane transducer that activates OmpR, a transcriptional activator for porin gene expression (ompC), in response to an aspartic acid signal. Signal transduction by Taz1 most likely involves a phosphorylated Taz1 intermediate that donates its phosphate to OmpR. Phosphorylated OmpR has already been implicated in transcriptional activation of porin genes. Using a cell-free system containing Taz1-enriched membrane fractions, we have examined the phosphorylation properties of Taz1 and the stimulatory effects of divalent and monovalent ions. Highest activation of Taz1 phosphorylation was observed with CaCl2, and its stimulation could be observed with as low as 60 microM of CaCl2. Phosphorylated Taz1 could readily donate its phosphate group to OmpR in the presence of calcium. CaCl2 was also able to enhance phosphorylation of intact membrane-bound EnvZ and a cytoplasmic fragment of EnvZ lacking the receptor and transmembrane domains. These results indicate that the site for CaCl2 stimulation is within the cytoplasmic region of EnvZ and probably involves an enhanced rate of EnvZ phosphorylation.
Rampersaud
A
A
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Utsumi
R
R
Delgado
J
J
Forst
S A
SA
Inouye
M
M
D001345:Autoradiography; D001419:Bacteria / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002628:Chemoreceptor Cells; D002678:Chimera; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009097:Multienzyme Complexes; D010766:Phosphorylation / Q000187:drug effects; D018272:Porins; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface; D015398:Signal Transduction
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D018272:Porins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D011494:Protein Kinases; C057843:envZ protein, E coli; D008274:Magnesium; D002118:Calcium