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.

Effects of the processing methods of corn grain and soybean meal on milk protein expression profiles in dairy cows

“…Expression of β-CN may have a compensation effect between the variants. The in vitro bovine mammary epithelial cell results indicated that not only did the cells grow but β-CN expression was also promoted with the addition of Lys to the media (Lu et al, 2013). Based on our findings, we hypothesize that concentrations of essential AAs, such as Lys, are higher in mammary gland cells because they can be supplemented by microbial protein in dairy cows fed SSBM, which displays a higher rumen degradable protein than HSBM.…”

Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells

“…To determine the effect of methionine (Met) on 14-3-3c, eIF1AX and RPS7 expression in BMECs, cells were treated with Met (0.6 mmol L À1 ) for 24 h as in our previous study [22]. Western blot and immunofluorescence analysis showed that 14-3-3c, eIF1AX and RPS7 were overexpressed after Met stimulation in BMECs ( Fig.…”

“…We carried out western blot analysis in the same way as previously reported [22]. Primary antibodies used in this work were: 14-3-3c (1:6000, Abcam, ab 79165, MA, USA), eIF1AX Densitometric analysis of the immunoreactive bands was performed using the ImageJ program (US National Institutes of Health, MA, USA).…”

Biosynthesis of milk fat, protein, and lactose: roles of transcriptional and posttranscriptional regulation

“…Leu affects translation by enhancing mTOR activity indirectly and the translation initiation complex directly. Lys appears to augment translation through the phosphorylation of Mitogen-activated Protein Kinase that in turn enhances the expression and phosphorylation of mTOR and STAT5a, as recently demonstrated in bovine mammary cells (133). Met is well known for its essential role in the initiation of mRNA translation, which is the binding of methionyl-tRNA to the 40S ribosomal subunit followed by the joining of a 60S ribosomal subunit to form a translationally competent 80S ribosome (112).…”