Authors: Tanoue T, Adachi M, Moriguchi T, Nishida E
Nat Cell Biol. 2000 Feb;2(2):110-116.

[1] Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto
606-8502, Japan.

Presenter: James Mwanjewe
 

Background: Mitogen-activated protein kinase (MAPK) pathways are protein kinase cascades that have a
function in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different signals and have a role in a wide variety of physiological processes. The best understood cascade is the MAPK-ERK pathway, a hierarchical cascade originating at the cell membrane with receptors for mitogens or growth factors, which recruit, via adapter proteins and exchange factors, the small guanosine triphosphatase (GTPase) Ras . Ras activates raf, a serine threonine kinase, which activates MEK (MAPK/ERK kinase). MEK, in turn, phosphorylates and activates ERK1 and ERK2, which translocate to the nucleus and transactivate transcription factors, changing gene expression to promote growth, differentiation or mitosis. Thus by transducing signals through a cascade of kinases, several options for control are introduced for amplifying and/or modifying the output signal.
MAPKs themselves are activated by Mitogen Activated Protein Kinase Kinases (MAPKKs), regulated by Mitogen Activated Protein Kinase Phosphatases (MKPs) and phosphorylate Mitogen Activated Protein Kinase Activated Protein Kinase (MAPKAPKs) as their substrate.

Experimental approach

They follow the following approach:

1. From the understanding that MAPKs form complex with their MAPKKs, MAPKAPKs, and MKPs, and transcription factors through docking sites, the group examined amino acid sequence of almost all known members of MAPKKs and MAPKAPKs.
2. They saw that all have a putative or identified MAPK docking site, characterized by a cluster of positively charged amino acids outside the catalytic domain where by at least two positively charged amino acids were together.
3. The same type of cluster was found in MKPs regulatory domains.
4. They then went on to the assumption that since the docking site for MAPKKs, MAPKAPKs, and MKPs have been found to have positively charged amino acids, the docking site on MAPKs is composed of negatively charged amino acids.
5. The group then hypothesized that for the MAPK- MEK2, its activator MEK1, its substrate MNK1 and regulator MKP3, all bind to the same site on ERK2. By using mutant constructs of ERK2, MEK1, MKP3 and MNK1, they were able to show that MEK1, MKP3 and MNK1 use their N terminal end, employing positive amino acids to dock on the carboxyl terminal of ERK2.
6. They then show that the docking site of ERK2 is indeed COMMON for MEK1, MKP3 and MNK1. They have termed the docking site on ERK2 as CD domain, (for Common Docking domain) in which two aspartic acids Asp 321 and Asp 324 are important for the docking.
7.  They also show that these docking interactions use electrostatic forces.
8. They also show that the docking interactions increase the efficiency of the enzymatic reactions of MAPKs.
9. Lastly they show that other members of the MAPK family, namely p38 and JNK also possess CD domains
 
 
 

Why this paper is important:
One the strong points of this paper the realization that formation of multimeric signaling complexes, as well as the dynamic movement of signaling proteins, contribute to determine signaling specificity and efficacy. The authors experimental approach is important, which leads them to show that MAPKs  indeed have a common binding site for their, substrates, activators and deactivators, thus bringing all these to close proximity, contributing to signal specificity.

Questions Arising:
1. Even though docking interactions increase the efficiency of the enzymatic reaction, the undocked mutant enzyme can phosphorylate Myelin Binding Protein (MBP), which means the enzyme does not undergo conformational change and the catalytic site is unaffected. So is increasing the specifity of the enzyme to its interacting molecules from the vast sea of other possible kinases, phosphatases and substrates the only importance of docking?
2. In the physiological state whereby the activators, regulators and substrate are present in equilibrium (rather than in the experimental state whereby one species is over-expressed), what criteria leads to one species(say the substrate)to dock to MAPK over the other species?
3. Since, activation, then phosphorylation of the substrate and eventually deactivation of MAPK presumably follow each other sequentially, what leads to the displacement of one moiety from MAPK by the others? Can say, the activators bind preferentially to the deactivators?

Abstract:

Mitogen-activated protein kinases (MAPKs) are specifically phosphorylated and activated by the MAPK kinases, phosphorylate various targets such as MAPK-activated protein kinases and transcription factors, and are inactivated by specific phosphatases. Recently, docking interactions via the non-catalytic regions of MAPKs have been suggested to be important in regulating these reactions. Here we identify docking sites in MAPKs and in MAPK-interacting enzymes. A docking domain in extracellular-signal-regulated kinase (ERK), a MAPK, serves as a common site for binding to the MAPK kinase MEK1, the MAPK-activated protein kinase MNK1 and the MAPK phosphatase MKP3. Two aspartic acids in this domain are essential for docking, one of which is mutated in the sevenmaker mutant of Drosophila ERK/Rolled. A corresponding domain in the MAPKs p38 and JNK/SAPK also serves as a common docking site for their MEKs, MAPK-activated protein kinases and MKPs. These docking interactions increase the efficiency of the enzymatic reactions. These findings reveal a hitherto unidentified docking motif in MAPKs that is used in common for recognition of their act