A crystallographic snapshot of tyrosine trans -phosphorylation in action

Abstract

Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme-and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 Å away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 transphosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.crystal structure ͉ FGF receptor ͉ RTKs S ignaling by receptor tyrosine kinases (RTKs) plays ubiquitous roles throughout the human life cycle commencing at germ cell maturation and continuing throughout embryogenesis into adulthood (1). Ligand binding to the extracellular region of RTKs triggers activation of the intracellular tyrosine kinase domain through the universal process of trans-phosphorylation, whereby one kinase acts as a substrate for another one. Trans-phosphorylation has two major roles in RTK signaling: trans-phosphorylation on A-loop tyrosines elevates enzyme activity while trans-phosphorylation of juxtamembrane and C-terminal tyrosines generate platforms for recruitment and phosphorylation of target substrates (2-6).Structural studies of tyrosine kinase domains have been instrumental in shaping our current understanding of the mechanisms of tyrosine kinase regulation. Crystal structures of unphosphorylated tyrosine kinase domains have unveiled diverse tactics used by kinases to achieve self-inhibition (7-10). The crystal structures of phosphorylated kinases, however, reveal how tyrosine phosphorylation stabilizes the active conformation of the kinase, and how peptide substrates dock into the enzyme active site (7,(11)(12)(13). In contrast, the structural basis for tyrosine trans-phosphorylation has remained elusive. Here, we report the crystal structure of a phosphorylated FGF receptor 2 (FGFR2) kinase domain, which provides the first crystallographic snapshot of trans-phosphorylation in action. Our structural data supported by biochemical data show that trans-phosphorylation proceeds with a much higher degree of specificity than that currently perceived based on the crystal structures of kinases in complexes with short peptide substrates. Results and DiscussionWe recently reported the crystal structure of the A-loop phosphory...

Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells

“…The N-lobe is rotated toward the C-lobe of the kinase structure as has previously been seen in activated phosphorylated FGFR structures (9,10). In the structure of FGFR1-RE, no density for ATP analog, ACP-PCP, was found in the catalytic cleft between the Nlobe and C-lobe.…”

“…Recently, an asymmetric dimer was described for the kinase domain of FGFR2 (9). In the FGFR2 crystal structure Y769 (equivalent to Y766 in FGFR1) is trapped in a position that seems poised to be a substrate for the other kinase domain.…”

“…Structural and biochemical studies have shown that autophosphorylation of fibroblast growth factor receptor 1 (FGFR1) (7,8) and FGFR2 (9) are mediated by a sequential and precisely ordered intermolecular reaction that can be divided into three phases. The first phase involves transphosphorylation of a tyrosine located in the activation loop (Y653 in FGFR1) of the catalytic core resulting in 50-100-fold stimulation of kinase activity (7).…”

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

“…However, the question as to why the triphosphate has adopted the observed uncommon and unproductive chelate conformation still remains. An analysis of the structures in the Protein Data Bank which contain an AMPPCP shows that while very few have been reported, most are complexes with kinase domains, either from FGF receptor 2 (FGFR2) (8,9) or human RSK-1 (24). Inspection of the bound AMPPCP in five FGFR2 structures shows that in all cases, the triphosphate adopts an extended conformation, interacting with two magnesium ions.…”

Plasticity in Interactions of Fibroblast Growth Factor 1 (FGF1) N Terminus with FGF Receptors Underlies Promiscuity of FGF1

“…The D1 and D1-D2 linker regions, albeit dispensable for ligand binding, are implicated in receptor autoinhibition because loss of these regions enhances the FGF and HS binding affinity of the D2-D3 region (35,36). Upon binding of ligand and HS, FGFRs assemble into a 2:2:2 FGF-FGFR-HS symmetric dimer (32,37,38) in which the juxtaposed cytoplasmic kinase domains gain sufficient opportu-nity to transphosphorylate each other on A-loop tyrosines and become activated (39,40). FGFR kinase activation triggers activation of various downstream signaling pathways, including the RAS-MAPK (41), PI hydrolysis/PKC/Ca 2ϩ (42,43), PI3K-AKT (44 -46), and RAC1/CDC42 (47) signaling pathways (48,49).…”