摘要Amino acids(AA),including methionine(Met)and leucine(Leu),stimulate milk synthesis in bovine mammary epithelial cells(BMEC)via activation of protein kinase mechanistic target of rapamycin(mTOR).In this study,we further explored the potential role of myotonic dystrophy-related CDC42-binding kinase alpha(MRCKα),previously identified as a critical mediator of prolactin-stimulated milk synthesis in BMEC.Administering different doses(0,0.2,0.4,0.6,0.8 mM)of Met or Leu to a primary BMEC culture showed that 0.6 mM was the optimal dose for stimulating β-casein production with both AA.At this dose,Met and Leu independently evoked higher(P＜0.05)protein levels of β-casein,MRCKαand sterol regulatory element-binding protein 1(SREBP1),and increased(P＜0.05)phosphorylation of mTOR(Ser2448)and phosphatidylinositol 3-kinase(PI3K)(Tyr317)after 24 h.The stimulatory effects of both AA on relative protein level of β-casein,phosphorylation of mTOR,and phosphorylation of protein kinase B(PKB)(Thr308),were blocked by silencing MRCKα expression(P＜0.05).Whereas,that on the phosphorylation of PI3K remained intact(P=0.385).Inhibiting PI3K with LY294002 blocked Met-and Leu-induced protein expression of MRCKα and β-casein and phosphorylation of mTOR(P＜0.05).Overexpression of MRCKα increased protein levels of β-casein and phosphorylation of mTOR,which was prevented by PKB inhibitor MK2206(P＜0.05).Our results indicate that MRCKα is a key mediator of the Met-and Leu-induced signaling cascade,acting downstream of PI3K and upstream of PKB to regulate β-casein synthesis in BMEC.