Leukemia Inhibitory Factor Activates Cardiac L-Type Ca 2+ Channels via Phosphorylation of Serine 1829 in the Rabbit Ca v 1.2 Subunit

Abstract

We have previously reported that leukemia inhibitory factor (LIF) gradually increased cardiac L-type Ca 2+ channel current ( I CaL ), which peaked at 15 minutes in both adult and neonatal rat cardiomyocytes, and this increase was blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. This study investigated the molecular basis of LIF-induced augmentation of I CaL in rodent cardiomyocytes. LIF induced phosphorylation of a serine residue in the α 1c subunit (Ca v 1.2) of L-type Ca 2+ channels in cultured rat cardiomyocytes, and this phosphorylation was inhibited by PD98059. When constructs encoding either a wild-type or a carboxyl-terminal–truncated rabbit Ca v 1.2 subunit were transfected into HEK293 cells, LIF induced phosphorylation of the resultant wild-type protein but not the mutant protein. Cotransfection of constitutively active mitogen-activated protein kinase kinase also resulted in phosphorylation of the Ca v 1.2 subunit in the absence of LIF stimulation. In in-gel kinase assays, extracellular signal–regulated kinase phosphorylated a glutathione S -transferase fusion protein of the carboxyl-terminal region of Ca v 1.2 (residues 1700 through 1923), which contains the consensus sequence Pro-Leu-Ser-Pro. A point mutation within this consensus sequence, which results in a substitution of alanine for serine at residue 1829 (S1829A), was sufficient to abolish the LIF-induced phosphorylation. LIF increased I CaL in HEK cells transfected with wild-type Ca v 1.2 but not with the mutated version. These results provide direct evidence that LIF phosphorylates the serine residue at position 1829 of the Ca v 1.2 subunit via the actions of extracellular signal–regulated kinase and that this phosphorylation increases I CaL in cardiomyocytes.