The study of MAPK and characterization of a novel ERK isoform in proliferative systems.

摘要

Mitogen-activated protein kinases (MAPK) form a family of protein kinases that are activated by numerous extracellular stimuli through complex networks of signaling cascades. These networks coordinately tranduce signals to many downstream serine/threonine kinases in a variety of cell types to achieve specific responses. Since MAPK play a central role in many signaling cascades in cell growth, proliferation, apoptosis, and differentiation it was of interest to examine the characteristics of MAPK activation in breast cancer. Examination of primary breast cancer tissue showed that several members of the MAPK family extracellular signal-regulated kinase 1 and 2 (ERK-1 and -2) were elevated in some 30 samples of breast cancer tissue. A distinct 45 kDa apparent ERK-4 isoform was also identified and found to be elevated in human breast cancer tissue as well. Interestingly there were also differences in the relative proportions of ERK-4, ERK-1, and ERK-2 within different breast tumor samples. The ERK-4 isoform was also found to be present in the cell cycle model system Xenopus Laevis oocytes and shown to share characteristics with the previously described ERK-4 from UT7 erythroleukemia cells.;Importantly ERK-4 is also abundant in breast cancer cell lines MDA-MB-468, MDA-MB-231, MDA-MB-435s, MDA-MB-361, SKBR3, MCF-7, and a normal breast cell line MCF-10 thus providing model systems for its further characterization. The 45 kDa ERK-4 is not a phosphorylated or activated form of the 44 kDa ERK-1, and has distinct characteristics demonstrated by modified SDS-PAGE, multi antibody immunoblot analysis, two-dimensional gel analysis, anion exchange chromatography, and hydrophobic interaction chromatography. However the ERK-4 protein does appear to be related to ERK-1 and ERK-2 based upon its immunoreactivity with antipeptide polyclonal ERK-1 antibodies that recognize the well characterized 42 kDa ERK-2 and 44 kDa ERK-1.;Finally, the effects of glucosamine and other modifiers of protein monoglycosylation were examined for their effects on ERK activation by epidermal growth factor (EGF) in breast cell lines MDA-MB-468 and SKRR-3. Interestingly, increasing protein monoglycosylation dampenes and shortens ERK-1 and ERK-2 activation by EGF in MDA-MB-468 cells, but increases and prolongs activity in SKBR-3 cells. A better understanding of the complex MAPK regulatory pathways including the unique ERK isoform distributions and their connections to protein monoglycosylation shown herein could lead to improved diagnosis and treatments for breast cancer.