ABSTRACT
Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.
Subject(s)
Aminopeptidases/metabolism , Cell Nucleus/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Serine Endopeptidases/metabolism , Aminopeptidases/antagonists & inhibitors , Animals , Cell Line , Cell Nucleus/drug effects , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , Gene Ontology , Humans , Inhibitory Concentration 50 , Isotope Labeling , Mice , Models, Biological , Neurites/drug effects , Neurites/metabolism , Neuronal Plasticity/drug effects , Phosphorylation/drug effects , Protein Phosphatase 2/metabolism , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism , SOXC Transcription Factors/genetics , SOXC Transcription Factors/metabolismABSTRACT
The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion. The overexpression of wild-type p27 or a phospho-mimetic p27S10D mutant in HepG2 cells induces a hyper-adhesive phenotype. In contrast, the overexpression of a nonphosphorylatable p27S10A mutant prevents the mobilization of E-cadherin and beta-catenin at the cell surface, reduces basal cell-cell adhesion strength, and prevents the stimulatory effect of oncostatin M on cell-cell adhesion. As part of the underlying molecular mechanism, it is shown that in p27S10A-expressing cells beta-catenin interacts with p27 and is prevented from interacting with E-cadherin. The intracellular retention of E-cadherin and beta-catenin is also observed in hepatocytes from p27S10A knockin mice that express the p27S10A mutant instead of wild-type p27. Together, these data suggest that the formation of adherens junctions in hepatocytes requires Ser-10 in p27.