ABSTRACT
p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.
Subject(s)
MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Nuclear Proteins/physiology , Signal Transduction , Animals , Apoptosis , Cell Differentiation , Cell Line , Cell Survival , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p57 , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Transfection , Ultraviolet RaysABSTRACT
SEDL (known also as sedlin) is a 140 amino-acid protein with a putative role in endoplasmic reticulum-to-Golgi transport. Several missense mutations and deletion mutations in the SEDL gene, which result in protein truncation by frame shift, are responsible for spondyloepiphyseal dysplasia tarda, a progressive skeletal disorder. The protein is identical to MIP-2A, which was shown to interact physically with c-myc promotor-binding protein 1 (MBP-1) and relieve the regulatory role of MBP-1 as a general transcription repressor. In order to gain insights into the function of SEDL by structural analysis, the protein was overexpressed and crystallized as a first step. SEDL was overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method at 298 K. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 46.69, b = 101.30, c = 66.15 A. The unit cell is likely to contain one molecule of SEDL, with a crystal volume per protein mass (V(M)) of 2.36 A(3)Da(-1) and a solvent content of about 47.9% by volume. A native data set to 2.8 A resolution was obtained from a flash-cooled crystal using synchrotron radiation.
