A genomic approach to biology is only meaningful when interpreted in a physiologic, functional context. Fortunately, we know a lot about the structure and function of both MEKs (reviewed by [17
]). MEK1 and MEK2 are dual specificity kinases that phosphorylate ERK1/2 at residues T202/185 and Y204/187 (reviewed by [18
]). The functional domains of human MEK1 and MEK2 are diagrammed in . Both MEK1 and MEK2 contain a central catalytic domain flanked on either side by short amino-terminal and carboxy-terminal regions and containing a proline-rich insert. These kinases are highly homologous, sharing 80% overall similarity, with 90% amino acid identity in their kinase domains. However, their sequences diverge in the flanking regions and the proline-rich insert. Each of these regions incorporates specific elements that distinguish these kinases, influencing interactions with other proteins and modifying kinase activity.
Figure 1: A linear model of MEK 1 and MEK 2 showing locations of identified functional regions and mutations. Linear models of MEK 1 and MEK 2 are used to indicate the locations of functional domains in both kinases. Above each is a scale with hash marks at the (more ...)
The amino-terminal flanking regions of MEK1 and MEK2 (amino acids 1–67 and 1–71, respectively; ) share 58% of the sequence identity. However, their sequences are less similar in the first 32 or 36 residues (MEK1 or MEK2, respectively) than they are in the remaining 28 or 24 residues (22% homology vs. 82%). This is notable because this least similar region mediates interaction with their ERK substrates. The ERK docking site or D-domain encompasses a short stretch of basic and hydrophobic amino acids located within the first 10 residues [19
]. The positively charged D-domain is essential for MEK binding to a complementary acidic common docking domain in the carboxy-termini of ERK1 and ERK2 [20
]. Deletion of the D-domain or mutation of either hydrophobic or basic residues reduced ERK phosphorylation [22
]. Similarly, proteolysis of the D-domain by anthrax lethal factor, the principle virulence factor secreted by B. anthracis
, inhibits MEK association with and activation of ERK [12
]. Genomic variants within this region may alter MEK:substrate interaction.
Between the D-domain and the catalytic core lies a nuclear export sequence (NES; amino acids 33–44 and 37–48; ). The NES is critical for MEK function and subcellular localization. MEKs normally reside in the cytoplasm. However, lysine to alanine substitution in the NES alters MEK steady-state cellular distribution to the cytoplasm and nucleus. This suggests MEKs translocate to the nucleus upon activation and then are rapidly exported to the cytoplasm in an NES-dependent manner [26
]. Genomic variants within this region may influence MEK subcellular localization.
Immediately following the NES lies a negative regulatory region (NRR; ). Deletion of residues 44–51 of MEK1 or 48–55 of MEK2 causes a 60- or 9-fold elevation in basal kinase activity, respectively [28
]. These residues repress MEK activity by forcing an inactive conformation that disrupts the ATP-binding site [29
]. Genomic variants within this region may alter MEK catalytic activity.
The proline-rich domain is located within the carboxy-terminal portion of the central conserved catalytic domain (amino acids 262–307 in MEK1 and 266–315 in MEK2; ) and is believed to mediate specific protein–protein interactions important for the regulation of the MEKs [30–32
]. Residues at the end of this segment have been reported to mediate binding with the bacterial virulence factors B. anthracis
] and Y. pestis
]. The proline-rich domains of MEK1 and MEK2 show 60% homology. Genomic variants within this region may influence MEK activity through altered protein complex formation.
The structure of the catalytic kinase core is broadly similar to that of other kinases and may be divided into a small amino-terminal lobe and a larger carboxy terminal lobe (). Located at the interface between these lobes are conserved regions playing important roles in ATP binding and hydrolysis, substrate recognition and phosphate transfer (reviewed by [17
]). Genomic variants within any of these regions may decrease the catalytic activity of MEK. Critical sites of this catalytic core include the glycine-rich P-loop (amino acids 74–82 of MEK1 or 78–86 of MEK2), the Mg2+
-positioning loop (amino acids 208–210 of MEK1 or 212–214 of MEK2), the ATP-binding site (amino acids 143–146 of MEK1 or 147–150 of MEK2), and the catalytic loop (amino acids 192–195 of MEK1 or 196–199 of MEK2).
The carboxy-terminal regions of MEK1 (amino acids 362–393) and MEK2 (amino acids 370–400) share 69% homology. Little is known of the function of this domain. However, Brunet et al.
] reported that ERK phosphorylation of MEK1 at T386 is a component of a negative feedback loop regulating MEK1 inactivation.
Additional insight into MEK structure and function comes from phosphorylation studies. Major phosphorylation sites at S218 and S220 of MEK1 or S222 and S226 of MEK2 are located within the activation segment (amino acids 208–233 of MEK1 or 212–237 of MEK2; , red hash marks with ovals). Phosphorylation at these sites causes a re-alignment of the loop that coordinates Mg2+
-positioning that is essential for ATP-binding and catalysis. Paradoxically, Gopalbhai et al.
] has reported phosphorylation at S212 in the activation loop reduces MEK1 activity. These reports indicate genomic variants within the activation loop may enhance or suppress the catalytic activity of MEK.
Phosphorylation outside the activation segment also influences MEK activity. PAK1-mediated phosphorylation of S298 in the proline-rich region is reported to induce MEK1 autophosphorylation at S218 and S222 [36
]. Elsewhere within the proline-rich domain MEK1 but not MEK2 has been reported to be phosphorylated on T286 and T292 and inactivated by cdc2 [38
]. In addition, Eblen et al.
] have shown that ERK2-mediated phosphorylation of MEK1 at T292 blocks PAK-dependent phosphorylation of S298. ERK-mediated phosphorylation of T292 has also been reported by Brunet et al.
]. In a recent study, Catalanotti et al.
] reported alanine substitution on MEK1 at T292 enhanced both MEK1 and MEK2 activation and ERK phosphorylation. Conversely, mutation of the same residue to aspartic acid dampened MEK1 and MEK2 activation and ERK phosphorylation. How this occurs is not known but the ability of MEK1 to modulate MEK2 activity may be related to the ability of these two kinases to form heterodimers because an N87G mutation in MEK1 blocks formation of heterodimers and prolongs growth factor stimulated activation of MEK2. These studies indicate genomic variants within the proline-rich region may enhance or suppress the MEK activity. Other phosphorylation sites have been identified in kinomic studies [41–44
] but the physiologic role of these is unknown (, red hash marks lacking ovals).