Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis

Abstract

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

Figure 1

Detection of cell viability and β-caseinsecretion of DCMECstreated with L-Lys. (A) Viability of cells at 24 h after adding L-Lys (0, 0.3, 0.6, 1.2, 2.4, 4.8 mmol L⁻¹); (B) Viability of cells at 0, 12, 24, 36, 48, 72 h after adding L-Lys1.2 mmol L⁻¹; (C) Changes of β-casein content of DCMECs at 12, 24, 36, 48, 72 h after adding L-Lys1.2 mmol L⁻¹. Values are means ± SE (n = 3 per group, biological replicates). * and ** indicate significant differences from values obtained in the control group at the same time point at levels of p < 0.05 and p < 0.01, respectively.

Figure 2

The nuclear phosphoproteomics of DCMECs treated with L-Lys. (A) Western blotting analysis showing the expression of markers in nuclear (Lamin B) and cytosolic (β-Tubulin) extracts of DCMECs; (B) Comparison of the gels of the control group with those of L-Lys-treated group. The differentially expressed proteins are indicated with arrows and numbered; (C) Close-up of the six differentially expressed proteins; (D) Comparative expression of genes as determined by RT-PCR. β-actin as control under the same condition. The ratio of the control group was regard as one; (E) Western blotting results of phospho-MAPK1, MAPK1 and β-actin; (F) Results of gray scale scan of phospho-MAPK1 and MAPK1.β-actin as control under the same condition. Values are means ± SE (n = 3 per group, biological replicates). * and ** indicate significant differences from values obtained in the control group at levels of p < 0.05 and p < 0.01, respectively.

Figure 3

MAPK1up-regulated expression of Stat5a and mTOR (A) Relative mRNA expression of MAPK1 was determined by qRT-PCR after MAPK1 inhibition; (B) Western blotting results of phospho-Stat5a, Stat5a, phospho-mTOR, mTOR and β-actin after MAPK1 inhibition; (C) Results of gray scale scan of phospho-mTOR, mTOR/β-actin relative fold by western blotting after MAPK1 inhibition; (D) Results of gray scale scan of phospho-Stat5a, Stat5a/ β-actin relative fold by western blotting after MAPK1 inhibition; (E) Relative mRNA expression of MAPK1 was determined by qRT-PCR after over-expression of MAPK1; (F) Western blotting results of phospho-Stat5a, Stat5a, phospho-mTOR, mTOR and β-actin after over-expression of MAPK1; (G) Results of gray scale scan of phospho-mTOR, mTOR/β-actin relative fold by western blotting after over-expression of MAPK1; (H) Results of gray scale scan of phospho-Stat5a, Stat5a/β-actin relative fold by western blotting after over-expression of MAPK1. Values are means ± SE (n = 3 per group, biological replicates). * and ** indicate significant differences from values obtained in the negative control group or the pGCMV-IRES-EGFP empty vector group at levels of p < 0.05 and p < 0.01, respectively.