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D Shalloway

Publications and source records attributed to D Shalloway.

At least 19 recordsLinked to original sources
PubMed2001

Two mechanisms activate PTPalpha during mitosis.

We show that, dependent on serine hyperphosphorylation, protein tyrosine phosphatase alpha (PTPalpha) is activated by two different mechanisms during mitosis: its specific activity increases and its inhibitory binding to Grb2 decreases. The latter effect probably abates Grb2 inhibition of the phosphotyrosine displacement process that is required specifically for Src dephosphorylation and causes a mitotic increase in transient PTPalpha-Src binding. Thus, part of the increased protein tyrosine phosphatase activity may be specific for Src family members. These effects cease along with Src activation when cells exit mitosis. Src is not activated in mitosis in PTPalpha-knockout cells, indicating a unique mitotic role for this phosphatase. The activation of PTPalpha, combined with the effects of mitotic Cdc2-mediated phosphorylations of Src, quantitatively accounts for the mitotic activation of Src, indicating that PTPalpha is the membrane-bound, serine phosphorylation-activated, protein tyrosine phosphatase that activates Src during mitosis.

3T3 Cells↗
PubMed2001

Top-down free-energy minimization on protein potential energy landscapes.

The hierarchical properties of potential energy landscapes have been used to gain insight into thermodynamic and kinetic properties of protein ensembles. It also may be possible to use them to direct computational searches for thermodynamically stable macroscopic states, i.e., computational protein folding. To this end, we have developed a top-down search procedure in which conformation space is recursively dissected according to the intrinsic hierarchical structure of a landscape's effective-energy barriers. This procedure generates an inverted tree similar to the disconnectivity graphs generated by local minima-clustering methods, but it fundamentally differs in the manner in which the portion of the tree that is to be computationally explored is selected. A key ingredient is a branch-selection algorithm that takes advantage of statistically predictive properties of the landscape to guide searches down the tree branches that are most likely to lead to the physically relevant macroscopic states. Using the computational folding of a beta-hairpin-forming peptide as an example, we show that such predictive properties indeed exist and can be used for structure prediction by free-energy global minimization.

Algorithms↗
PubMed2001

Qmol: a program for molecular visualization on Windows-based PCs.

With the advent of powerful, inexpensive graphics hardware, a variety of molecular modelling tools are now available for personal computers (PCs). While a host of high-quality, free visualization programs exist for Unix-like operating systems, there are relatively few available for Microsoft Windows. Most programs that do run under Windows are designed to interactively visualize experimental data and cannot read binary trajectory data generated by molecular simulations. In addition, many of these programs do not accept command line arguments, limiting their ability to serve as a "helper" application to be run by other applications. To fill this gap in functionality, we present Qmol--a program designed for viewing the output of simulations and theoretical calculations of peptides and small molecules.

Chemistry Techniques, Analytical↗
PubMed2000

A phosphotyrosine displacement mechanism for activation of Src by PTPalpha.

Protein tyrosine phosphatase alpha (PTPalpha) is believed to dephosphorylate physiologically the Src proto-oncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPalpha overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPalpha is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPalpha activity. We show here that this phosphorylation also specifically enables PTPalpha to dephosphorylate pTyr527. Tyr789-->Phe mutation abrogates PTPalpha-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPalpha in vivo. We suggest that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, we find that excess SH2 domains can disrupt PTPalpha-Src binding and can block PTPalpha-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, we show that, as predicted by the model, catalytically defective PTPalpha has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways.

Animals↗
PubMed2000

Shadow mass and the relationship between velocity and momentum in symplectic numerical integration.

It is often assumed, when interpreting the discrete trajectory computed by a symplectic numerical integrator of Hamilton's equations in Cartesian coordinates, that velocity is equal to the momentum divided by the physical mass. However, the "shadow Hamiltonian" which is almost exactly solved by the symplectic integrator will, in general, induce a nonlinear relationship between velocity and momentum. For the (symplectic) momentum- and midpoint-momentum-Verlet algorithms, the "shadow mass" that relates velocity and momentum is momentum independent only for a quadratic potential and, even in this case, differs from the physical mass. Thus, naively assuming the standard velocity-momentum relationship leads to inconsistencies and unnecessarily inaccurate estimates of velocity-dependent quantities. As examples, we calculate the shadow Hamiltonians for the momentum- and midpoint-momentum-Verlet solutions of the multidimensional harmonic oscillator, and show how their velocity-momentum relationships depend on the time step. Of practical importance is the conclusion that, to gain the full advantage of symplecticity, velocities derived from interpolated positions, rather than conventional velocity-Verlet velocities, should be used to compute physical properties.

Algorithms↗
PubMed1999

Characterization of Raf-1 activation in mitosis.

We have used site-directed mutagenesis to explore the mechanisms underlying Raf-1 activation in mitosis, and we have excluded most previously characterized activating interactions. Our results indicate that the primary locus of activation lies in the carboxyl-half of the molecule, although the extent of activation can be influenced by the amino-proximal region, particularly by the Raf-1 zinc finger. We also found that Raf-1 is hyperphosphorylated in mitosis at multiple sites within residues 283-302 and that these hyperphosphorylations are not required for activation. In addition, neither Mek1 nor Mek2 are stably activated in coordination with Raf-1 in nocodazole-arrested cells. Overall, the data suggest that the mechanism(s) responsible for activating Raf-1 during mitosis, and the subsequent downstream effects, are distinct from those involved in growth factor stimulation.

14-3-3 Proteins↗
PubMed1998

Specific correlations between relative synonymous codon usage and protein secondary structure.

We found significant species-specific correlations between the use of two synonymous codons and protein secondary structure units by comparing the three-dimensional structures of human and Escherichia coli proteins with their mRNA sequences. The correlations are not explained by codon-context, expression level, GC/AU content, or positional effects. The E. coli correlation is between Asn AAC and the C-terminal regions of beta-sheet segments; it may result from selection for translational accuracy, suggesting the hypothesis that downstream Asn residues are important for beta-sheet formation. The correlation in human proteins is between Asp GAU and the N termini of alpha-helices; it may be important for eukaryote-specific sequential, cotranslational folding. The kingdom-specific correlations may reflect kingdom-specific differences in translational mechanisms. The correlations may help identify residues that are important for secondary structure formation, be useful in secondary structure prediction algorithms, and have implications for recombinant gene expression.

Bacterial Proteins↗
PubMed1998

KH domain integrity is required for wild-type localization of Sam68.

The protein Sam68 (Src-associated in mitosis, 68 kDa) has been found to bind to SH2 and to SH3 domain-containing proteins and to RNA. Although its protein-protein interactions implicate Sam68 in cell signaling, the significance of its RNA binding remains obscure. In most cells, Sam68 shows diffuse nucleoplasmic staining. Upon treatment with transcription inhibitors, however, Sam68 localize into punctate nuclear structures. Mutant forms of mouse Sam68 were overexpressed in human cells to test the importance of the KH domain, which is required for RNA binding, in the intracellular localization of Sam68. A small deletion within the KH domain (delta 206-218) or point mutation I184N had no effect upon the localization of overexpressed Sam68. Sam68 that contained a deletion of the entire KH domain (delta KH, delta 157-256) or point mutation G178E, however, localized to distinct nuclear spots. Furthermore, delta KH Sam68, unlike wild-type Sam68 and several other mutant Sam68 proteins, did not relocalize upon poliovirus infection and caused the normally cytoplasmic viral polymerase to localize to the nuclear spots. Thus both ongoing transcription and an intact KH domain are crucial determinants of the dynamic intracellular localization of Sam68.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains.

Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain. To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. Deletion of the KH domain or mutation of KH domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1 abolished G8-5 binding. Our results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Association between v-Src and protein kinase C delta in v-Src-transformed fibroblasts.

In response to the kinase activity of v-Src there is an increase in the membrane association of the novel protein kinase C (PKC) isoform PKC delta (Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367-1373). We report here that in v-Src-transformed cells PKC delta co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC delta had reduced enzymatic activity relative to the non-tyrosine-phosphorylated PKC delta from v-Src-transformed cells. The association between Src and PKC delta was dependent upon both an active Src kinase and membrane association. The association between c-Src Y527F and PKC delta was substantially enhanced by mutating a PKC phosphorylation site at Ser-12 in Src to Ala indicating that PKC delta phosphorylation of Src at Ser-12 destabilizes the interaction, possibly in a negative feedback loop. These data demonstrate that upon recruitment of PKC delta to the membrane in v-Src-transformed cells there is the formation of a Src.PKC delta complex in which PKC delta becomes phosphorylated on tyrosine and down-regulated.

Animals↗
PubMed1997

Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis.

Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein which associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. We now show that Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues exclusively during mitosis as well as on serine residues during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, was phosphorylated on threonine and serine residues after incubation with mitotic lysates several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis.

3T3 Cells↗
PubMed1996

Cell cycle-dependent activation of Ras.

BACKGROUND: Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS: To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS: Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.

3T3 Cells↗
PubMed1996

Src and the control of cell division.

The finely tuned mechanisms that control cell cycle progression go awry in cancer, pointing to proto-oncogene products as important players in cell-cycle regulation. One such proto-oncoprotein, c-Src, has previously been directly implicated, based on its requirement for growth factor-stimulated DNA synthesis. Roche et al. have now shown that c-Src or its close relatives are also required for cell division to occur. The demonstration of essential functions for the Src family at multiple points in the cell cycle raises important questions about the normal and transforming activities of these and other proto-oncoproteins.

Animals↗
PubMed1995

Raf-1 is activated during mitosis.

The Raf-1 serine/threonine kinase transmits proliferative and developmental signals to downstream effectors in the mitogen-activated protein kinase cascade. Studies to date have concentrated on its role in growth factor-mediated activation of the cell cycle. We show here that Raf-1 kinase activity is also highly stimulated during mitosis, with an attendant distinctive electrophoretic mobility shift due to hyperphosphorylation. These results suggest that Raf-1 may play a role in traversal of at least two distinct phases of the cell cycle.

3T3 Cells↗
PubMed1995

Autophosphorylation of purified c-Src at its primary negative regulation site.

The phosphorylating and transforming activities of c-Src are negatively regulated by phosphorylation at Tyr-527 near its carboxyl terminus. Previous studies have indicated that c-Src preferentially autophosphorylates Tyr-416, a residue in the middle of the catalytic domain, in vitro, and that Tyr-527 is phosphorylated by the carboxyl-terminal Src kinase, Csk. However, indirect evidence suggests that c-Src may also autophosphorylate Tyr-527 as part of a negative feedback loop. While some in vivo evidence suggests that Tyr-527 can be autophosphorylated in an intermolecular interaction, it has not previously been possible to directly demonstrate significant autophosphorylation in vitro. Here we show that c-Src purified from recombinant bacteria can autophosphorylate Tyr-527 to high levels in vitro when incubated with sufficiently high concentrations of ATP (KM(Mg2+/ATP) approximately equal to 20 microM) that are well above those that have been used previously. In vitro Tyr-527 autophosphorylation can occur both as an intra- and intermolecular interaction; higher enzyme concentrations are required for intermolecular Tyr-527 phosphorylation than for Tyr-416 autophosphorylation. These results support the possibility that, like G-proteins, c-Src can switch itself off in vivo by its own enzymatic activity.

Escherichia coli↗
PubMed1995

Functional interaction between c-Src and its mitotic target, Sam 68.

The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src approximately Ras GTPase activating protein > p85 alpha (amino-terminal) > Grb2 >> p85 alpha (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 alpha SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68.

3T3 Cells↗
PubMed1995

Transformation of mouse cells by wild-type mouse c-Src.

Previous studies in which chicken and human c-Src were overexpressed in chicken and rodent cells have indicated that overexpression of wild-type c-Src can not induce complete neoplastic transformation. However, studies with v-Src mutants have demonstrated that species-specific differences can play a significant role in transforming activity. Here we show that, in contrast to chicken c-Src, overexpressed mouse c-Src can induce significant anchorage-independent growth and tumorigenicity when transfected into NIH3T3 mouse cells. The biochemical cause for this difference is unknown. In particular, the protein-tyrosine kinase activities of chicken and mouse c-Src appear to be similar. This result is consistent with the hypothesis that v-Src-induced transformation results from perturbation of signalling pathways modulated by c-Src and highlights the need for caution in controlling for potential species-specific differences in studies of c-Src function.

Animals↗