We have previously characterized the MEKK1 protein kinase as an upstream mediator of the Stress Activated Protein Kinase cascade. Stress Activated Protein Kinase (SAPK, alternatively known as JNK) cascade has recently been closely investigated owing to its participation in many physiological and pathologic stimuli, particularly inflammatory cytokines. Also of central importance is the yet unclarified role of MEKK1 in carcinogenesis. The Stress-Activated cascade is stimulated by DNA damaging agents that lead to programmed cell death (apoptosis) and are commonly used in chemotherapeutic strategies. MEKK1 and SAPK are broadly assumed to play a role in the apoptotic response, though our recent data suggest that effects of MEKK1 on apoptosis are complex and can be either positive or negative towards cell death. Central questions in signaling through MEKK1 are 1) its means of activation, and 2) its mechanism of transmitting the """"""""stress signal"""""""" through to cell growth machinery. MEKK1 is a 200 kD protein with a large amino terminal regulatory domain that has uncharacterized function. While MEKK1 is phosphorylated within the catalytic domain, our recent results indicate that phosphorylation is not the major mechanism of regulation of MEKK1 function. We have instead obtained recent evidence that two highly unusual mechanisms regulate MEKK1 functions. The first of these is targeted proteolysis of MEKK1. We have identified three distinct proteolytic cleavage sites on MEKK1, and hypothesize that different stimulatory events activate MEKK1 in functionally distinguishable ways. During apoptosis, proteases termed caspases cleave MEKK1 and redirect its activity away from the anti-apoptotic NFkappaB pathway, and instead promote activation of the SAPK-mediated AP1 transcription factor pathway. Secondly, we have identified reactive quinones as potent activators or inhibitors of the SAPK pathway (depending on concentration) and the two electron quinone reductase NQO1 as critical for transmission of stress signaling pathways upstream of MEKK1. We present data leading to the conclusion that regulation of sulfhydryl redox status is a prime means of MEKK1 activation, and hypothesize that MEKK1 itself is a target for redox regulation via its amino terminus. Considering the downstream function of stress signaling, we have found that the SAPK protein kinase becomes activated during cell cycle transition, during G2/M. Additionally, expression of MEKK1 or treatment of cells with various stress agents leads to inhibition of CDK kinase activity associated with cyclin B. We propose to characterize the mechanism by which stress signals interact with the cyclin B/cdc2 kinase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA066134-05
Application #
6172615
Study Section
Pathology B Study Section (PTHB)
Program Officer
Blair, Donald G
Project Start
1995-09-01
Project End
2004-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
5
Fiscal Year
2000
Total Cost
$280,626
Indirect Cost
Name
Case Western Reserve University
Department
Pathology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Cross, Janet V; Templeton, Dennis J (2004) Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain. Biochem J 381:675-83
Zhang, Donna D; Lo, Shih-Ching; Cross, Janet V et al. (2004) Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol 24:10941-53
Seanor, Krista L; Cross, Janet V; Nguyen, Suzanne M et al. (2003) Reactive quinones differentially regulate SAPK/JNK and p38/mHOG stress kinases. Antioxid Redox Signal 5:103-13
Cross, J V; Deak, J C; Rich, E A et al. (1999) Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis. J Biol Chem 274:31150-4
Deak, J C; Cross, J V; Lewis, M et al. (1998) Fas-induced proteolytic activation and intracellular redistribution of the stress-signaling kinase MEKK1. Proc Natl Acad Sci U S A 95:5595-600
Deak, J C; Templeton, D J (1997) Regulation of the activity of MEK kinase 1 (MEKK1) by autophosphorylation within the kinase activation domain. Biochem J 322 ( Pt 1):185-92