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Abstract
MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.
Highlights
We have cloned four MEK kinases (MEKKs),1 referred to as MEKK1, -2, -3, and -4 [1,2,3]
We have demonstrated that the 113-kDa fragment B is the result of cleavage of MEKK1 by caspase-3 at Asp-874, which generates a 91-kDa C-terminal activated kinase domain
With respect to 14-3-3 interactions, these results indicate that the kinase domains of MEKK1, -2, and -3 are inherently different, even though their amino acid sequence is conserved
Summary
(Received for publication, September 16, 1997, and in revised form, November 18, 1997). Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins. Caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins. Relevant to the regulation of signal transduction pathways, 14-3-3 proteins have been shown to interact with Raf-1, Bcr-Abl, polyoma middle tumor antigen, KSR (kinase suppressor of Ras), the Bcl family member BAD, the platelet adhesion receptor, glycoprotein Ib-IX, insulin receptor substrate-1, and protein-tyrosine phosphatase H1 (14 –17, 19, 21–24). We demonstrate that caspase-3 cleavage of MEKK1 releases the active kinase domain from the N-terminal 14-3-3-binding region, defining a new function for caspases, namely the release of active kinase from its cellular scaffold
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