Biomedical subjects

D R Stover

Publications and source records attributed to D R Stover.

10 recordsLinked to original sources

PubMed2004

Effect of polymorphisms on the replicative capacity of protease inhibitor-resistant HIV-1 variants under drug pressure.

The role of drug pressure on the replicative capacity of protease inhibitor-resistant HIV-1 variants and the contribution of a common amino-acid polymorphism in the protease gene (L63P) to this process were investigated. Using HIV-1 variants resistant to the protease inhibitors saquinavir (G48V/L90M) or indinavir (A71V/V82T/I84V), viral replication was studied in the presence or absence of inhibitor and a mutation at position 63. The initial changes diminished enzyme function of the protease and reduced replicative capacity for both virus mutants. Addition of the respective inhibitor blocked the wild-type, but was also able to delay the replication kinetics of either mutant, revealing the limits of resistance. Importantly, the polymorphic change L63P, although not conferring inhibitor resistance by itself, provided a significant replication benefit to both mutant viruses, particularly under drug pressure, and may reveal a far-reaching compensating power of polymorphic changes. This may drive evolution and the directed selection of protease inhibitor-resistant HIV-1 variants, a finding with significant clinical and diagnostic implications.

Amino Acid Sequence↗

PubMed1999

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.

Amino Acid Sequence↗

PubMed1999

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

Adenosine Triphosphate↗

PubMed1998

One-step purification of cathepsin D by affinity chromatography using immobilized propeptide sequences.

In vivo, active cathepsin D proteinase is generated by removal of a 44-residue propeptide at its N-terminus. Here we report that mature cathepsin D and pseudocathepsin D (a partially activated form of cathepsin D with 25 amino acid residues removed from the propeptide) bind to the immobilized propeptide, while procathepsin D does not. The N-terminal 25 amino acid residues of the propeptide are sufficient for this binding. Based on this observation, a simple one-step procedure was developed to purify mature cathepsin D from whole cell extracts to near homogeneity. This method has the advantage over existing affinity-purification systems that active forms of the proteinase can be separated from inactive precursors and other aspartic proteinases. Furthermore, this technique was effective for pepsin as well, suggesting it may have general utility for all activated aspartic proteinases and perhaps other families of proteinases.

Amino Acid Sequence↗

PubMed1996

Modulation of the SH2 binding specificity and kinase activity of Src by tyrosine phosphorylation within its SH2 domain.

The Src family of kinases are held in an inactive state by interaction of their SH2 domain with a C-terminal phosphotyrosine. Dephosphorylation of this site can reactivate Src; however, recent evidence suggests that activation can also occur without dephosphorylation. In this study, platelet-derived growth factor receptor phosphorylation of Src on Tyr-213 specifically blocked binding of its SH2 domain to a phosphopeptide corresponding to the C-terminal regulatory sequence, while binding to other sequences, such as the platelet-derived growth factor receptor or a peptide from the epidermal growth factor receptor, was unaffected. Consequently, Src was activated over 50-fold. This is the first demonstration of regulation of a SH2 domain specificity by post-translational modification and is likely to be a general mechanism for regulation of all Src-like kinases.

Amino Acid Sequence↗

PubMed1995

Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and P85 alpha.

Following ligand binding, the epidermal growth factor receptor (EGF-R) autophosphorylates itself on tyrosine residues located in its carboxyl terminus; in vitro, three sites are highly phosphorylated, while two other sites are phosphorylated to lesser extents. In the presence of the Src protein-tyrosine kinase, in vitro phosphorylation of the minor autophosphorylation sites was increased, and four additional residues were phosphorylated. Following EGF stimulation, two (Tyr-891 and Tyr-920) were found to be phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor cell line (MCF7). The remaining in vitro sites were not found to be highly phosphorylated in vivo. The sequences surrounding Tyr-891 and Tyr-920 match the reported consensus binding sequences for the SH2 domains of Src and the regulatory domain of phosphatidylinositol 3-kinase (p85 alpha), respectively. In vitro, both of these proteins were found to bind to Src-phosphorylated EGF-R with approximately 100-fold greater affinity than to autophosphorylated EGF-R, demonstrating that Src creates new sites for SH2 binding. Furthermore, Csk-inactivated Src was activated by interaction with Src-phosphorylated EGF-R but not by autophosphorylated EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with both p85 alpha and Src. Evidence is also presented that suggests that an EGF-R-related protein, ErbB2, may be involved in similar Src-mediated interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel sites can act as docking sites for Src, P85 alpha, and potentially other SH2-containing proteins. In addition, the data suggest a tyrosine phosphatase-independent mechanism for the elevation of Src activity in cells exposed to growth factors. Overexpression of Src, EGF-R, and/or ErbB2 in breast and colorectal tumor cells suggests the potential that such interactions may contribute to the transformed phenotype of these carcinomas.

Amino Acid Sequence↗

PubMed1994

Cdc2-mediated modulation of pp60c-src activity.

Following complete dephosphorylation, purified p60c-src was rephosphorylated with Csk. The Csk-phosphorylated form was isolated and found to be essentially inactive. Both the dephosphorylated p60c-src (Src A) and the inactive, phosphorylated pp60c-src (Src B) were then used to explore the regulatory role of other kinases and phosphatases. Phosphorylation by Cdc2 partially reactivated Csk-inactivated pp60c-src. This reactivation occurred in the absence of Tyr-527 dephosphorylation. Moreover, phosphorylation of Csk-treated pp60c-src by Cdc2 also facilitated complete reactivation by the protein-tyrosine phosphatase CD45 or by a synthetic phosphopeptide corresponding to the C-terminal, regulatory phosphorylation site (Tyr-527). These data indicate that the Src homology 2 domain of Csk-phosphorylated pp60c-src was more accessible for intermolecular interactions and that Tyr-527 was more readily dephosphorylated after treatment with Cdc2. In conjunction with in vivo studies, these data suggest that Cdc2 is involved in the regulation of pp60c-src during mitosis.

Amino Acid Sequence↗

PubMed1994

Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.

Animals↗

PubMed1993

Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45.

It has been reported that alteration of deletion of critical residues within one of the two homologous protein tyrosine phosphatase (PTPase)-like domains of CD45 completely abolishes all activity, suggesting that only the more N-terminal domain is catalytically active. However, we now demonstrate, by two independent techniques, that the second (C-terminal) domain is also a viable phosphatase. Limited proteolysis by endoproteinase Lys-C or trypsin increased the phosphatase activity toward reduced, carboxymethylated, and maleylated lysozyme approximately 8-fold. A 50-kDa fragment, isolated by ion exchange chromatography, was found to be responsible for this activity. N-terminal sequencing revealed that this fragment includes less than half of the first phosphatase domain and most, if not all, of the second. In a second experiment, 109 residues, including the presumed catalytic region, were removed from domain I by site-directed mutagenesis. Expression of this construct in a mammalian cell line resulted in increased PTPase activity over nontransfected control cells. Isolation of the recombinant CD45 by immunoprecipitation and immunoaffinity chromatography revealed that it had phosphatase activity. Both of these experimental approaches demonstrate that the second conserved PTPase domain of CD45 is a functioning PTPase, but that external regulation may be required to express its activity in the context of the native molecule.

Blotting, Western↗

PubMed1991

Protein-tyrosine-phosphatase CD45 is phosphorylated transiently on tyrosine upon activation of Jurkat T cells.

The leukocyte common antigen (CD45) is an abundant lymphocyte surface antigen that has been reported to be involved in signaling through the T-cell antigen receptor. CD45 is a transmembrane protein-tyrosine-phosphatase. An internal segment comprises two domains each of which is homologous to other protein-tyrosine-phosphatases; the extracellular segment has the hallmarks of a ligand-binding motif. Since tyrosine phosphorylation is an early signal resulting from stimulation of the T-cell antigen receptor and CD45 is required for proper activation through the receptor, we explored whether CD45 might be regulated by tyrosine phosphorylation. Treatment of a T-cell leukemia line (Jurkat) with either phytohemagglutinin or anti-CD3 antibodies induced phosphorylation of tyrosine residues in CD45; treatment with phorbol 12-myristate 13-acetate did not. Phosphorylation of CD45 was transient, disappearing within 40 min after phytohemagglutinin treatment. The requirement for stringent conditions of phosphatase inhibition suggests that CD45 is capable of autodephosphorylation in vivo. These observations support recent reports indicating CD45 is involved in an early step in the T-cell activation cascade. They also suggest that phosphorylation/dephosphorylation of tyrosine residues in CD45 should be explored further as a possible regulatory mechanism.

Antigens, CD↗