Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

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 (<i>P</i> < 0.05) protein levels of β-casein, MRCKα and sterol regulatory element-binding protein 1 (SREBP1), and increased (<i>P</i> < 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 (<i>P</i> < 0.05). Whereas, that on the phosphorylation of PI3K remained intact (<i>P</i> = 0.385). Inhibiting PI3K with LY294002 blocked Met- and Leu-induced protein expression of MRCKα and β-casein and phosphorylation of mTOR (<i>P</i> < 0.05). Overexpression of MRCKα increased protein levels of β-casein and phosphorylation of mTOR, which was prevented by PKB inhibitor MK2206 (<i>P</i> < 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.