Proteomic Analysis of Nuclear Phosphorylated Proteins in Dairy Cow Mammary Epithelial Cells Treated with Prolactin

Prolactin (PRL) is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells (DCMECs), nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry (MALDI-TOF MS) were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying≥2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS (GlyRS), which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1 (Heat shock protein 47), which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions, such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species (ROS). ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2+-dependent phospholipid-binding protein, maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several cellular processes, including cell cycle control, cellular stress response, intracellular signaling. This screening revealed that prolactin influenced the level of nuclear phosphoproteins in DCMECs. This result opens new avenues for the study of the molecular mechanism linked to the synthesis of milk proteins.