PARENT SESSION
PLATFORM SESSION 11: MOLECULAR MECHANISMS OF HORMONE ACTION
Chair: Nilson, John¹, ¹
Co-chair: Horvat, Regina¹, ¹
Grand Ballroom I-IV
2:00 PM-4:00 PM

(231) AG1478 SENSITIVE TYROSINE KINASE AND CALCIUM ARE INVOLVED IN OXYTOCIN-STIMULATED ERK1/2 PHOSPHORYLATION.

Zhong, Miao¹, Yang, Ming¹, Sanborn, Barbara¹, ¹ Department of Biochemistry & Molecular Biology, Houston, TX

ABSTRACT- Both Gq-PLC-PKC and pertussis toxin-sensitive Gi pathways have been implicated in oxytocin (OT)-stimulated ERK1/2 activation, and tyrosine kinases have been implicated in OT-stimulated gene activation. Some pathways link G-protein coupled receptors to ERK1/2 activation via transactivation of EGF receptor (EGFR). To examine the role of tyrosine kinases in OT-stimulated ERK1/2 activation, plasmid expressing OT receptor (0.5 g) was transiently transfected into COSM6 cells using lipofectamine, and the phosphorylation of ERK1/2 (ERK1/2-P) was determined in immunoblots of whole cell extracts with an anti-P-ERK1/2 antibody. Data were normalized to total ERK1/2. OT-stimulated (10nM, 5 min) ERK1/2-P (3.4±0.5-fold over control, n=9) was completely blocked by preincubation with the OT antagonist vasotocin (1 M, 1h). Preincubation of the cells with the EGFR tyrosine kinase inhibitor AG1478 (0.1 M, 1h) markedly reduced OT-induced ERK1/2-P (83%±4%, n=3), whereas the PDGF receptor tyrosine kinase inhibitor AG1296 (1 M) had no effect. AG1478 did not affect IP₃ production by 40 nM OT, suggesting that OT stimulation of PLC lies upstream of or parallel to the AG1478-sensitive pathway. PKC activation lies downstream of PLC. Activation of PKC with PMA (1 M, 5min) also stimulated ERK1/2-P (2.7±0.3 fold, n=3) but was not affected by pretreatment with AG1478. The PKC inhibitor Go6983 (100 nM) only slightly (<30%) reduced OT-induced ERK1/2-P. Extracellular EGTA (0.2 mM) reduced OT-induced ERK1/2-P by 58%±8% (n=3); no ERK1/2-P was observed when intracellular increases in Ca²⁺ were blocked by 0.1 M thapsigargin plus extracellular EGTA. These data suggest that an AG1478-sensitive tyrosine kinase, possibly EGFR, and Ca²⁺ play important roles in OT receptor-stimulated, PKC-independent ERK1/2 phosphorylation. Supported by HD09618.

KEY WORDS: oxytocin receptor, calcium, AG1478, PKC