La formación: una práctica que ejerce tensiones en la configuración del maestro

The activation of all RTKs follows some general rules. Ligand binding brings at least two catalytically repressed kinase-domains together [9,105], which phosphorylate each other in trans. Phosphorylation of the kinase activation loop leads to the exposure of the kinase-active site, which is usually blocked. In some RTKs like Eph, KIT, Flt3 (vascular endothelial growth factor receptor 3), PDGFβ (platelet derived growth factor β) and TrkB, the juxtamembrane region is also involved in regulation of the kinase activity [106,107]. In Ephs, catalytic activation correlates with phosphorylation of two tyrosines (Tyr604 and Tyr610, for murine EphB2) within the JM segment [108-110], and possibly the phosphorylation of a third tyrosine in the kinase activation segment (referred to as TyrAct788, for murine EphB2;

TyrAct779 for EphA4). In vivo phosphorylation sites were reported to be Tyr667, Tyr744 and

Tyr750 [111], however, their role for Eph receptor regulation is so far unknown.

Fig. 1.4 Molecular mechanism of Eph receptor kinase activation.

Schematic presentation of the proposed activation mechanism by means of EphB2/EphA4 structural studies [107,112]. For better understanding of the structural features in relation to EphB2 sequence alignment, an illustration is shown on the bottom. (A) Unphosphorylated, autoinhibited state: the N- terminal lobe elements implicated in nucleotide binding are well ordered and adopt a prototypical

protein kinase arrangement except for obvious distortions in helix αC and the G-loop. The G-loop

plays an important role in coupling the β-strand movements to produce an altered twist to that of helix

αC (indicated tachometer symbol). This overall structural scenario results from interactions with the JM

segment (black arrows). In the C-lobe of the kinase domain, the activation segment, which is also located in the large catalytic lobe (not depicted here), is disordered (indicated by intensely scribbled light-blue line) and produces a steric contact with Tyr750 (indicated by red scribbled line), which adopts an alternate conformation impeding the activation segment from adopting a stabilizing path.

The preceding JM segment is highly ordered forming αA’ and αB’ helices with intimate contact to αC

helix of the N-terminal kinase lobe (black arrows) and limited interactions of the C-terminal lobe (black

stippled arrow). This leads to an imposed kink on helix αC. The distortion couples to distortions of the

N-terminal lobe elements, which appear to impinge on catalytic function by adversely affecting the coordination of the sugar and phosphate groups of the bound nucleotide (not depicted here). (B) Phosphorylated, active state: phosphorylation of Tyr604 and Tyr610 serves to destabilize the JM structure through electrostatic repulsion exerted by negatively charged phosphate groups. This

abrogates the intimate contact to helix αC and the C-terminal lobe to cause overall enhanced interlobe

dynamics comprising helix αC and the activation segment. Tyr750 adopts a conformation that no

longer impedes the productive ordering of the activation loop. The dynamic conformational equilibrium

is shifted from a kinked to a straight αC helix allowing the return of the N-terminal lobe to an

undistorted active conformation. Conformational rearrangements are mediated via the G-loop to

produce an altered twist in β-strand secondary structures (indicated by arrow in circle). The current

idea is that catalysis is followed by dynamic fluctuations from a more stable conformation favoring a rather dynamic picture of Eph receptor kinase activation over the transition to a static active state. Red arrows indicate structural interactions.

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G-loop JM segment

N-terminal kinase C-terminal kinase lobe

αC

αB’

αA’ β1

β2-3 β4-5

Y604 Y610

Y750 activation segment 712

622 638-641

features

/aa residue (EphB2)

Tyr610

Tyr604 Tyr750

kinked

αC

restriction of interlobe motion

activation segment N-lobe β-sheet contact G-loop JM segment

sampling of straight helixαC conformation G-loop SH2 SH2 αC N-lobe β-sheet straight αC activation segment Tyr750 JM segment Tyr604 Tyr610 αB’ αA’ contact

A

B

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X-ray crystallography of the juxtamembrane and kinase domains of EphB2 indicate a dual inhibition mechanism involving the kinase activation loop and the two conserved juxtamembrane tyrosine residues [107]. In its non-phosphorylated form, the JM segment folds back to form a well-ordered structure, interacting with the N-terminal lobe of the kinase, presumably causing the distortion of the key α-helix C leading to repression of the kinase activity (Fig. 1.4A). Interestingly, α-helix C possesses a kink not previously observed in active state protein kinase structures. In addition, limiting the JM segment contacts with the kinase domain C-lobe appear to prevent the activation segment from adopting an ordered active conformation. The JM segment bridges the N- and C-lobes of the kinase and thereby restricts inter-lobe flexibility. Together, these disruptive features are suggested to account for the repression of kinase activity of Eph receptors in their autoinhibited states. Upon phosphorylation of the JM tyrosines electrostatic repulsion would then lead to a relief of the structural constrains controlling kinase activity [107]. Mutation of these JM tyrosines to negatively charged glutamate (eeEph) in EphA4 leads to a constitutively kinase-active EphA4 receptor, which remains sensitive to receptor ligation in vivo [113]. Exchange for uncharged phenylalanine (ffEph), in contrast, leads to a receptor frozen in its autoinhibited state [107]. Moreover, phosphorylation of the JM tyrosines enables docking of SH2-binding adaptor proteins (SH2 - Src homology domain 2) inducing further downstream signaling events [114]. Recently, the Sicheri lab performed a much more detailed study of Eph kinase activation utilizing a combination of mutational and structural analyses, also including NMR spectroscopy, to draw a more dynamic picture of autoinhibited and active forms of EphB2 and EphA4 [112]. They provided direct evidence that phosphorylation of the JM segment residues Tyr604 and Tyr610 and a gain-of-function point mutation of Tyr750 to alanine in the C-lobe of the kinase domain induces disorder of the JM segment and its dissociation from the kinase domain (Fig. 1.4B).

Interestingly, these induced disorders in the JM segment occur without major conformational changes to the kinase domain and with only partial ordering of the kinase domain activation segment. All these results suggest that rather a change in interlobe dynamics of the JM segment and kinase domain, than a transition to a static active conformation forms the mechanistic basis for Eph RTK activation [112].

While the importance of JM tyrosine phosphorylation for Eph receptor activation is well established, phosphorylation of other residues, such as Tyr788, Tyr667, Tyr744 and Tyr750 also seem to influence the stability of the autoinhibited structure and hence Eph receptor activity. [107,112].

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