Insights into the Structural Basis of the GADD45β-mediated Inactivation of the JNK Kinase, MKK7/JNKK2

Salvatore Papa,Rosa Maria Vitale,Concetta Bubici,Enrico De Smaele,Guido Franzoso,Carlo Pedone,Nina Dathan,Simona M Monti,Menotti Ruvo,Kellean Alvarez,Shanthi Jayawardena

doi:10.1074/jbc.m703112200

Abstract

NF-kappaB/Rel factors control programmed cell death (PCD), and this control is crucial to oncogenesis, cancer chemoresistance, and antagonism of tumor necrosis factor (TNF) alpha-induced killing. With TNFalpha, NF-kappaB-mediated protection involves suppression of the c-Jun-N-terminal kinase (JNK) cascade, and we have identified Gadd45beta, a member of the Gadd45 family, as a pivotal effector of this activity of NF-kappaB. Inhibition of TNFalpha-induced JNK signaling by Gadd45beta depends on direct targeting of the JNK kinase, MKK7/JNKK2. The mechanism by which Gadd45beta blunts MKK7, however, is unknown. Here we show that Gadd45beta is a structured protein with a predicted four-stranded beta-sheet core, five alpha-helices, and two acidic loops. Association of Gadd45beta with MKK7 involves a network of interactions mediated by its putative helices alpha3 and alpha4 and loops 1 and 2. Whereas alpha3 appears to primarily mediate docking to MKK7, loop 1 and alpha4-loop 2 seemingly afford kinase inactivation by engaging the ATP-binding site and causing conformational changes that impede catalytic function. These data provide a basis for Gadd45beta-mediated blockade of MKK7, and ultimately, TNFalpha-induced PCD. They also have important implications for treatment of widespread diseases.

Highlights

Tumor progression [2, 3]
We and others have shown that, downstream of tumor necrosis factor (TNF)-Rs, the NF-␬B-mediated antagonism of programmed cell death (PCD) involves a suppression of the sustained activation of the c-Jun N-terminal kinase (JNK) cascade [6, 7]
These regions appear to establish several bonds with residues in the catalytic pocket and the ␤3-helix C loop of MKK7, preventing access of the kinase to ATP and inducing conformational changes that stabilize this kinase in an inactive conformation

Summary

EXPERIMENTAL PROCEDURES

Protein Purification and Binding Assays—Glutathione S-transferase (GST) proteins used Figs. 1– 4 were purified from bacterial lysates using glutathione beads (Sigma) as detailed elsewhere [11]. Protein Purification and Binding Assays—Glutathione S-transferase (GST) proteins used Figs. 1– 4 were purified from bacterial lysates using glutathione beads (Sigma) as detailed elsewhere [11]. In vitro translated proteins (5 ␮l) were incubated with GST (10 ␮l) proteins for 20 min and pulled down with glutathione beads as described previously [11]. Additional Methods—A detailed description of additional materials and methods, including cell treatments and death assays, plasmids, human (h)Gadd45␤ purification, native gel electrophoresis, gel filtration, LC-MS, circular dichroism (CD), limited proteolysis and alkylation analyses, and modeling can be found in the supplementary data

RESULTS

To tighten the link between

DISCUSSION

Ruvo and Guido Franzoso