Characterization and Kinetic Mechanism of Catalytic Domain of Human Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase (VEGFR2 TK), a Key Enzyme in Angiogenesis

No thorough mechanistic study of extracellular signal-regulated protein kinase 2 (ERK2) has appeared in the literature. A recombinant protein termed Ets⌬138, which comprises of residues 1-138 of the transcription factor Ets-1 is an excellent substrate of ERK2 (Waas W. F., and Dalby, K. N. (2001) Protein Exp. Purif. 23,[191][192][193][194][195][196][197]. The kinetic mechanism of ERK2 was examined, with excess magnesium, by initial velocity measurements, both in the absence and presence of products at 27°C, pH 7.5, and ionic strength 0.1 M (KCl). The velocity data are consistent with a steady-state randomordered ternary complex mechanism, where both substrates have unhindered access to binding sites on the enzyme. The mechanism and magnitude of product inhibition by monophosphorylated Ets⌬138 is consistent with, but does not prove, the notion that ERK2 forms a discrete interaction with Ets⌬138 in the absence of active site interactions, and that this "docking complex" facilitates intramolecular phosphorylation of the substrate. The approximation of the steady-state data to a rapid equilibrium model strongly suggests that the formation of ERK2⅐Ets138 complexes are transient in nature with dissociation constants of greater magnitude than the catalytic constant, of k cat ‫؍‬ 17 s ؊1 .Extracellular regulated protein kinase 2 (ERK2) 1 is a prominent, ubiquitously expressed, 42-kDa cellular protein kinase, which is strongly activated by phorbol esters, growth factors, and serum (1). It is also activated, albeit more weakly, by a diverse range of general and cell type-specific stimuli, such as cell stresses, various cytokines, and insulin (1). ERK2 is some 85% identical in sequence (44 kDa) to ERK1 (2), which displays a similar profile of activation, activity, and expression. Both are associated with a myriad of biological processes, which include exit of cells from G 0 into the G 1 stage of the cell cycle, proliferation, mammalian synaptic plasticity and learning, glycogen metabolism, and many events that are often specific to certain tissues (3).ERK2 is activated through a protein kinase cascade termed the mitogen-activated protein kinase (MAPK) pathway, usually by Ras, a small guanine nucleotide-binding protein that transduces extracellular signals to the cell nucleus. Mutations in the ras oncogene family are commonly found in both human and animal cancers and in particular tumors of the lung, thyroid, pancreas, colon, and some leukemias (4). When unregulated, Ras signaling can lead to the development of both primary tumors and metastases through the sustained action of downstream effectors such as ERK2. Events leading to the activation of the ERKs appear to be highly orchestrated, but while many proteins have been shown to interact with the major protagonists of the MAPK pathway the precise functions of protein-protein interactions within it are poorly understood (5).Defining and understanding protein-protein interactions mediated by MAP kinases has far reaching consequences for both the design of inhibitors and for...

Section: Discussionmentioning

confidence: 99%

Transient Protein-Protein Interactions and a Random-ordered Kinetic Mechanism for the Phosphorylation of a Transcription Factor by Extracellular-regulated Protein Kinase 2

Waas

Dalby

2002

“…For cAMPdependent kinase Whitehouse et al (50,51) reported that the mechanism was ordered, with the nucleotide binding first while Cook et al (30) reported that MgATP and peptide bind randomly, although initial binding of the nucleotide is preferred. An ordered sequential mechanism has been reported for p38 MAPK (32), for the vascular endothelial growth factor receptor-2 tyrosine kinase (52) and for the v-Src kinase (53). Both an ordered (54) and a random pathway (55) have been reported for the EGF receptor tyrosine kinase.…”

Section: Table II Summary Of Kinetic Constantsmentioning

confidence: 95%

The Cyclin-dependent Kinases cdk2 and cdk5 Act by a Random, Anticooperative Kinetic Mechanism

Clare¹,

Poorman²,

Kelley³

et al. 2001

cdk2⅐cyclin E and cdk5⅐p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2⅐GST-cyclin E and cdk5⅐GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K i value of 0.6 mM. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K i value of 2 M. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2⅐GST-cyclin E the kinetic parameters were determined to be K m, ATP ‫؍‬ 3.6 ؎ 1.0 M, K m, peptide ‫؍‬ 4.6 ؎ 1.4 M, and the anticooperativity factor, ␣ ‫؍‬ 130 ؎ 44. For cdk5⅐GST-p25, the K m, ATP ‫؍‬ 3.2 ؎ 0.7 M, K m, peptide ‫؍‬ 1.6 ؎ 0.3 M, and ␣ ‫؍‬ 7.2 ؎ 1.8.

“…Biochemical characterization of RTKs has been performed using both recombinant truncated kinase domains and cytoplasmic domains (5,(11)(12)(13)(14). Cheng and Koland (14) showed that the cytoplasmic domain of the EGF receptor has an almost 10-fold greater K d for an ATP analog than a carboxyl terminus deletion mutant of the cytoplasmic tail (14), demonstrating that the isolated kinase domain may not be a sufficient model for receptor function.…”

Section: Stimulation Of Receptor Tyrosine Kinases (Rtks)mentioning

confidence: 99%

“…Studies with polyclonal antibodies have qualitatively shown that a phosphorylated monomeric receptor does not possess the ability to phosphorylate exogenous substrate in the same manner as the oligomeric receptor (8). All of these studies would indicate that there is some unique functional characteristic contained within the oligomeric RTK that is not present in the monomeric receptor.Biochemical characterization of RTKs has been performed using both recombinant truncated kinase domains and cytoplasmic domains (5,(11)(12)(13)(14). Cheng and Koland (14) showed that the cytoplasmic domain of the EGF receptor has an almost 10-fold greater K d for an ATP analog than a carboxyl terminus deletion mutant of the cytoplasmic tail (14), demonstrating that the isolated kinase domain may not be a sufficient model for receptor function.…”

mentioning

confidence: 99%

Oligomerization-induced Modulation of TPR-MET Tyrosine Kinase Activity

Hays¹,

Watowich

2003

Phosphorylation, although necessary, may not be sufficient to fully activate many receptor tyrosine kinases (RTKs). Oligomerization-induced conformational changes may be necessary to modulate the kinetic properties of RTKs and render them fully functional. To investigate this regulatory mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative of the RTK c-MET, has been expressed and purified for quantitative enzymatic analysis. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR). cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified for comparative enzymatic analysis. ATP and peptide substrates have been kinetically characterized for both TPR-MET and cyto-MET. Significantly, phosphorylated TPR-MET has smaller K m values for ATP (K m,ATP ) and peptide substrates (K m,peptide ) and a larger k cat relative to phosphorylated cytoMET. This provides the first direct evidence that receptor oligomerization and not simply activation loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation constants (K d,ATP ) for the two enzymes also displayed significant differences. In contrast, the K I values for the ATP competitive inhibitor staurosporin are similar for the two phosphorylated enzymes. These results suggest that much of the oligomerizationinduced kinetic changes occur with respect to peptide substrate binding or catalytic efficiency. The possibility that oligomerization-induced conformational changes occur within the cytoplasmic domain of receptor tyrosine kinases has significant implications for structure-based design of RTK inhibitors and the development of a detailed mechanistic model of RTK activation. Stimulation of receptor tyrosine kinases (RTKs)1 by ligand binding initiates an intracellular signaling cascade that can induce cellular differentiation, proliferation, and/or migration, among other cellular responses (1). Binding of a cognate extracellular ligand to a monomeric RTK induces receptor oligomerization and, in turn, autophosphorylation of tyrosine residues within the cytoplasmic tail of the receptor (1-3). The phosphorylated form of the receptor recruits signaling molecules through Src homology 2 or phosphotyrosine binding domains, and subsequent activation of these molecules initiates intracellular signaling cascades (1, 4).The molecular details of how RTK oligomerization regulates its function are incompletely understood. It is known that receptor oligomerization enhances autophosphorylation of tyrosine residues within the kinase regulatory domain and that this phosphorylation results in localized conformational changes that increase the kinase activity of the receptor (5-7). However, regulatory domain phosphorylation, although necessary, may not be sufficient to fully activate the kinase (8, 9). Additional oligomerization-induced conformational changes may be necessary to increase the kinase catalytic activity and render the RTK fully functional. E...