Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy.

Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.

Structural basis of oncogenic activation caused by point mutations in the kinase domain of the MET proto-oncogene: modeling studies.

Missense mutations in the tyrosine kinase domain of the MET proto-oncogene occur in selected cases of papillary renal carcinoma. In biochemical and biological assays, these mutations produced constitutive activation of the MET kinase and led to tumor formation in nude mice. Some mutations caused transformation of NIH 3T3 cells. To elucidate the mechanism of ligand-independent MET kinase activation by point mutations, we constructed several 3D models of the wild-type and mutated MET catalytic core domains. Analysis of these structures showed that some mutations (e.g., V1110I, Y1248H/D/C, M1268T) directly alter contacts between residues from the activation loop in its inhibitory conformation and those from the main body of the catalytic domain; others (e.g., M1149T, L1213V) increase flexibility at the critical points of the tertiary structure and facilitate subdomain movements. Mutation D1246N plays a role in stabilizing the active form of the enzyme. Mutation M1268T affects the S+1 and S+3 substrate-binding pockets. Models implicate that although these changes do not compromise the affinity toward the C-terminal autophosphorylation site of the MET protein, they allow for binding of the substrate for the c-Abl tyrosine kinase. We provide biochemical data supporting this observation. Mutation L1213V affects the conformation of Tyr1212 in the active form of MET. Several somatic mutations are clustered at the surface of the catalytic domain in close vicinity of the probable location of the MET C-terminal docking site for cytoplasmic effectors.

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site.

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.

Addressing the issue of sequence-to-structure alignments in comparative modeling of CASP3 target proteins.

During a blind protein structure prediction experiment (the third round of the Critical Assessment of Techniques for Protein Structure Prediction; URL http://PredictionCenter.llnl.gov/casp3/) , four target proteins, T0047, T0048, T0055, and T0070, were modeled by comparison. These proteins display 62%, 29%, 24%, and 19% sequence identity, respectively, to the structurally homologous proteins most similar in sequence. The issue of sequence-to-structure alignment in cases of low sequence homology was the main emphasis. Selection of alignments was made by constructing and evaluating three-dimensional models based on series of samples produced mainly by automatic multiple sequence alignments. Sequence-to-structure alignments were correct in all but two regions, in which significant changes in target structures compared with related proteins were the source of errors. Template choice is an important determinant of model quality, and a correct selection was made of a lower homology template for modeling of T0070; however, in the case of T0055, a template with 8% greater sequence homology proved deceptive. Loops and some ungapped template regions were assigned conformations taken from other proteins. Using fragments from homologous structures led to improvement over template backbone more often than cases in which nonhomologous structures were the source. The results also indicate that side-chain prediction accuracy depends not only on sequence similarity but also on accuracy of the backbone.

Modelling of insulin receptor tyrosine kinase in its active form: a case study for validation of theoretical methods.

An active form of an insulin receptor tyrosine kinase (IRK) catalytic core was modelled based on its experimentally known inactive form and the active form of a serine/threonine kinase, protein kinase A (PKA). This theoretical model was compared with the crystallographic structure of the active form of IRK reported later. The structures are very similar, which shows that all the most important features and interactions have been taken into account in the modelling procedure. The elaborated procedure can be applied to other tyrosine kinases. This would allow designing of a wide class of tyrosine kinase inhibitors, very important potential anti-cancer and/or anti-viral drugs.

Modelling of active forms of protein kinases: p38--a case study.

An active form of p38 protein kinase, belonging to the mitogen-activated protein kinases subfamily, has been designed based on crystallographically known structures of two other kinases, an active form of protein kinase A (PKA) and an inactive form of extracellular signal-regulated kinase 2 (ERK2). The modelling procedure is described. Its general scheme can also be applied to other kinases. The structure of the active forms of p38 and PKA is very similar in the region which binds the substrate. The ATP-binding mode is very similar in the active forms of all the three studied kinases. Models of the active forms allow for further studies on transphosphorylation processes at the molecular level, and modelling of inhibitors competitive with ATP and/or substrates.