Biomedical subjects

Kui Shen

Publications and source records attributed to Kui Shen.

13 recordsLinked to original sources

PubMed2006

An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor.

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.

Allosteric Regulation↗

PubMed2006

The role of the phospho-CDK2/cyclin A recruitment site in substrate recognition.

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 A from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the gamma-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 microm) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

Adenosine Triphosphate↗

PubMed2006

A Src-like inactive conformation in the abl tyrosine kinase domain.

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

Amino Acid Motifs↗

PubMed2005

Protein kinase structure and function analysis with chemical tools.

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase-substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.

Animals↗

PubMed2004

Surgical treatment of nonfunctioning islet cell tumor: report of 41 cases.

BACKGROUND: Nonfunctioning islet cell tumor (NIT) as a rare pancreatic endocrine neoplasm is characterized by unspecific clinical symptoms and is hard to diagnose. In China, NIT accounts for 15%-41% in pancreatic endocrine neoplasms just next to insulinoma. In this study, we evaluated the surgical modalities of NIT. METHODS: From January 1978 through February 2002, 41 patients with NIT were treated at the Department of Surgery of the First Affiliated Hospital, China Medical University, Shenyang, China. Tumors in the head of the pancreas were noted in 28 patients, and in the body or in the tail in 13 patients. The mean diameter of the tumors was 10.7 cm. Fifteen patients underwent enucleation and 21 received pancreatectomy. Tumors were unresectable in 5 patients because of extensive infiltration. The mean diameter was 9.6 cm in patients treated by enucleation, 13.1 cm in those by pancreaticoduodenectomy, 9.9 cm in those by distal pancreatectomy, and 11.6 cm in those with unresectable tumors. RESULTS: The curative resection rate was 88% (n=36), and the complication rate after enucleation and pancreatectomy was 33% (n=5) and 14% (n=3), respectively. No local recurrence was found after both enucleation and pancreatectomy. Liver metastases occurred in 3 patients treated by enucleation. CONCLUSIONS: Both enucleation and pancreatectomy are effective for NIT of the pancreas. No local recurrence has been found in patients treated by the two surgical procedures. The complication rates of the two modalities are comparable.

Adenoma, Islet Cell↗

PubMed2003

Conversion of a tyrosine kinase protein substrate to a high affinity ligand by ATP linkage.

Protein kinases often show low affinity for their protein substrates, which makes it difficult to study kinase-substrate interactions. Here, we show using expressed protein ligation with the signaling protein Src that it is feasible to install a covalently linked ATP moiety into the tail of Src, generating a semisynthetic protein with a high affinity for its cognate tyrosine kinase, Csk. It is also established that this Src-ATP conjugate can be used to selectively pull down Csk from a complex protein mixture. This work outlines a general strategy for identifying an unknown kinase that is responsible for the phosphorylation of a protein substrate on a site of interest.

Adenosine Triphosphate↗

PubMed2003

Cellular effects of small molecule PTP1B inhibitors on insulin signaling.

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type 2 diabetes and other associated metabolic syndromes. To further define the role of PTP1B in insulin signaling and to test the hypothesis that blocking the activity of PTP1B would augment the action of insulin, we prepared several cell permeable, potent and selective, small molecule PTP1B inhibitors, and evaluated their biological effects in several insulin sensitive cell lines. Our data indicate that PTP1B inhibitors bind to and colocalize with PTP1B on the surface of the endoplasmic reticulum and PTP1B exerts its negative effect on insulin signaling upstream of phosphatidylinositol 3-kinase and MEK1. Treatment of cells with PTP1B inhibitors, both in the presence and in the absence of insulin, markedly enhances IRbeta and IRS-1 phosphorylation, Akt and ERK1/2 activation, Glut4 translocation, glucose uptake, and Elk1 transcriptional activation and cell proliferation. These results indicate that small molecule inhibitors targeted to PTP1B can act as both insulin mimetics and insulin sensitizers. Taken together, our findings combined with results from PTP1B knockout, antisense, and biochemical studies provide strong evidence that PTP1B negatively regulates insulin signaling and that small molecule PTP1B inhibitors have the ability to potentiate and augment the action of insulin.

Animals↗

PubMed2003

Chemical dissection of the effects of tyrosine phosphorylation of SHP-2.

The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.

Amino Acid Sequence↗

PubMed2003

Crystal structure of PTP1B complexed with a potent and selective bidentate inhibitor.

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-A resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.

Alkanesulfonic Acids↗

PubMed2003

Protein tyrosine kinases Src and Csk: a tail's tale.

Csk and Src are two protein tyrosine kinases with similar amino acid sequences but very different structures and functions. Csk catalyzes a tail phosphorylation reaction on Src and thereby restrains Src's activity and oncogenic potential. Comparative analysis of the domain interactions in these enzymes provides a lesson in signalling diversity and mechanisms of enzyme regulation. The molecular basis of the specificity of Csk targeting the Src tail appears to involve both local and long-range interactions and illustrates the complexity of selective targeting in post-translational modification.

Animals↗

PubMed2003

Chemical approaches to reversible protein phosphorylation.

Protein phosphorylation catalyzed by protein kinases plays a critical role in cellular signaling. Here we review several chemical approaches to understanding protein kinases and the consequences of protein phosphorylation. We discuss the design of bisubstrate analogue inhibitors based on a dissociative transition state, the development of reagents for cross-linking protein kinases with their substrates, the chemical rescue of mutant protein tyrosine kinases, and the application of expressed protein ligation to understanding protein phosphorylation.

Amino Acid Sequence↗

PubMed2002

The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation.

The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.

Escherichia coli↗

PubMed2002

Probing the molecular basis for potent and selective protein-tyrosine phosphatase 1B inhibition.

Protein-tyrosine phosphatases (PTPs) are important for the control of proper cellular tyrosine phosphorylation. Despite the large number of PTPs encoded in the human genome and the emerging roles played by PTPs in human diseases, a detailed understanding of the role played by PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific inhibitors. Such inhibitors could serve as useful tools for determining the physiological functions of PTPs and may constitute valuable therapeutics in the treatment of several human diseases. However, because of the highly conserved nature of the active site, it has been difficult to develop selective PTP inhibitors. By taking an approach to tether together two small ligands that can interact simultaneously with the active site and a unique proximal noncatalytic site, we have recently acquired Compound 2 (see Fig. 1), the most potent and selective PTP1B inhibitor identified to date, which exhibits several orders of magnitude selectivity in favor of PTP1B against a panel of PTPs. We describe an evaluation of the interaction between 2 and its analogs with PTP1B and its site-directed mutants selected based on hydrogen/deuterium exchange of PTP1B backbone amides in the presence and absence of 2. We have established the binding mode of Compound 2 and identified 12 PTP1B residues that are important for the potency and selectivity of Compound 2. Although many of the residues important for Compound 2 binding are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which suggest that the binding surface defined by these residues in individual PTPs determines inhibitor selectivity. Our results provide structural information toward understanding of the molecular basis for potent and selective PTP1B inhibition and further establish the feasibility of acquiring potent, yet highly selective, PTP inhibitory agents.

Amino Acid Sequence↗