The cardiac hypertrophic factors modulate the function of the cardiac L-type Ca channels

白血病抑制因子（LIF）是IL-6细胞因子家族的一员，可诱导心肌肥厚。本研究小组报道了LIF可以激活被PD98059 （MEK抑制剂）阻断的心脏l型Ca^<2+>通道。我们还发现，LIF与活化的ERK、CaMK-IV和钙调磷酸酶一起诱导心肌肥厚，这依赖于通过l型Ca^<2+>增加的Ca^<2+>电流。为了确定LIF是否磷酸化α1亚基，α1亚基是l型Ca^<2+>通道功能的主要调节因子，用LIF刺激新生大鼠心肌细胞原代培养15分钟，并以重组al亚基（含有800个羧基末端氨基酸的gst融合蛋白）为底物，通过凝胶激酶法测定激酶活性。ERK1/2磷酸化了含有ERK1/2基序特定丝氨酸的融合蛋白。LIF促进了免疫沉淀α1亚基的磷酸化，其代谢标记为更多的h P-32。磷酸化氨基酸分析表明，LIF仅在丝氨酸位置磷酸化al亚基。将兔α1亚基靶丝氨酸的点突变体转染HEK293细胞。LIF显著增强了转染野生型α1亚基的磷酸化，而突变体则没有。将野生型α1亚基与组成活性MEK1共转染HEK293细胞后，用P-32标记α1亚基，其磷酸化水平显著提高。通过穿孔斑块-团块法，含有兔野生型α1亚基的l型Ca^<2+>通道在LIP给药后Ca^<2+>电流延长，而点突变体的通道则没有延长。这些发现表明，LIF在体外和体内均可磷酸化心脏l型Ca^<2+>通道α1亚基的靶丝氨酸，因此ERK1/2可能是心脏l型Ca^<2+>通道的潜在激活剂。少

The leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family that can induce cardiac hypertrophy. Our group reported that LIF can activate cardiac L-type Ca^<2+> channels blocked by PD98059 (MEK inhibitor). We also showed that LIF induces cardiac hypertrophy in concert with activated ERK, CaMK-IV and calcineurin, which are dependent on increased Ca^<2+> currents through L-type Ca^<2+>. To determine whether LIF phosphorylates the α1 subunits, the main regulator of the channel function of L-type Ca^<2+> channels, primary cultures of neonatal rat cardiomyocytes were stimulated with LIF for 15 min, and the kinase activity measured by the in-gel-kinase assay using recombinant al subunits (GST-fusion proteins containing 800 amino acids of the carboxyl terminal) as the substrate. ERK1/2 phosphorylated the fusion protein containing the certain serine of the ERK1/2 motif. LIF promoted the phosphorylation of the immuno-precipitated α1 subunits, which was metabolically labeled wit … More h P-32, significantly. Phospho-amino acid analysis showed that LIF phosphorylated the al subunits only at the serine position. The point mutant of the target serine of the rabbit α1 subunit was transfected into HEK293 cells. LIF enhanced phosphorylation of the transfected wild-type α1 subunit by significantly, but not that of the mutant. The wild-type α1 subunits which were co-transfected into HEK293 cells with the constitutively active MEK1 were labeled with P-32, and theses were showed enhanced phosphorylation significantly. By the perforated patch-clump method, the L-type Ca^<2+> channels containing the rabbit wild-type α1 subunit showed the prolongation of the Ca^<2+> current after LIP administration, but the channels of the point mutant did not showed the prolongation. These findings indicate that LIF phosphorylates the target serine of the α1 subunit of cardiac L-type Ca^<2+> channels both in vitro and in vivo, and that ERK1/2 may therefore be a potential activator of cardiac L-type Ca^<2+> channels. Less