Multiple signals regulate phospholipase CBeta3 in human myometrial cells.
Phospholipase CB3 (PLCB3) serine(1105) (S(1105)), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S(1105) phosphorylation (P-S(1105)) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S(1105). Relaxin also stimulated P-S(1105); this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S(1105), as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S(1105), whereas the reverse was the case for the oxytocin-stimulated increase in P-S(1105). PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S(1105)A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S(1105) dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.
Zhong
Miao
M
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Murtazina
Dilyara A
DA
Phillips
Jennifer
J
Ku
Chun-Ying
CY
Sanborn
Barbara M
BM
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002116:Calcitonin / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D025461:Feedback, Physiological; D005260:Female; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009215:Myometrium / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010121:Oxytocin / Q000494:pharmacology; D054799:Phospholipase C beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011498:Protein Precursors / Q000494:pharmacology; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction
C000606589:CALCA protein, human; D017931:DNA Primers; D011498:Protein Precursors; D011994:Recombinant Proteins; D012694:Serine; D010121:Oxytocin; D002116:Calcitonin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C518039:PLCB3 protein, human; D054799:Phospholipase C beta; D015740:Calcitonin Gene-Related Peptide