Vol. 300, Issue 1, 339-345, January 2002

Extracellular Signal-Regulated Kinases and G Protein-Coupled Receptors in Megakaryocytic Human Erythroleukemia Cells: Selective Activation, Differential Regulation, and Dissociation from Mitogenesis

Hung Wu, Huang-Wei Shen, Tin-Feng Wu, Lawrence F. Brass and Kuo-Chun Sung

Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan (H.W., H.-W.S., T.-F.W., K.-C.S.); and Departments of Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (L.F.B.)

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are a group of kinases that play an important role in proliferation and differentiation. In megakaryocyte-like human erythroleukemia (HEL) cells, ERK2 was found to be predominantly expressed and strongly activated by prostaglandin (PG) E₂, thrombin, and epinephrine. On the other hand, adenosine, ADP, ATP, and UTP did not significantly increase ERK1/2 phosphorylation. However, of the agonists tested, only ADP was able to stimulate thymidine uptake. Pretreatment with pertussis toxin abolished the PGE₂ response but had less of an effect on thrombin. PGE₂- and thrombin-induced ERK1/2 activation was mimicked by 4--phorbol-12-myristate-13-acetate and ionomycin and blocked by mitogen-activated protein kinase kinase inhibitor 1,4 diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene but displayed differential sensitivity to protein kinase C inhibitor bisindolylmaleimide I and Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Analogs of cAMP or agents that stimulate cAMP production were either weak or ineffective activators. Further studies indicate that the effect of thrombin was blocked by the phosphoinositide 3-kinase inhibitor wortmannin but not by agents inhibiting tyrosine kinase activity. On the contrary, herbimycin, but not wortmannin, attenuated the effect of PGE₂. Collectively, these results indicate that ERK1/2 are selectively activated by G protein-coupled receptors and not functionally associated with proliferation in HEL cells. ERK1/2 activation in response to PGE₂ and thrombin is mediated by distinctive types of G proteins and is differentially regulated by multiple pathways, including calcium mobilization, protein kinase C, phosphoinositide 3-kinase, and tyrosine kinases.