Biomedical subjects

D Shalloway

Publications and source records attributed to D Shalloway.

At least 19 recordsLinked to original sources

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↗

PubMed1994

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Base Sequence↗

PubMed1994

An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis.

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

3T3 Cells↗

PubMed1994

Differential effect of phosphorylation and substrate modulation on tau's ability to promote microtubule growth and nucleation.

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. To study the effect of phosphorylation and substrate modulation on tau's distinct activities to promote growth of existing microtubules and nucleation of new ones, we phosphorylated bacterially expressed human tau by cAMP-dependent protein kinase in the absence or presence of heparin, an acidic substrate modulator. We found that heparin increased phosphorylation of tau by a factor of more than 2 and produced tau bands with decreased electrophoretic mobility. We demonstrate that phosphorylation of tau in the absence or presence of heparin similarly reduced tau's activity to promote microtubule growth, whereas tau's activity to promote microtubules was suppressed much more after phosphorylation in the presence of heparin. Using recombinant tau fragments we showed that heparin-induced phosphorylation caused a specific shift in electrophoretic mobility indicative of a change in tau's conformation. By aminoterminal sequencing of a tau fragment starting at residue 154 we provide evidence that phosphorylation of serine 156 is responsible for this mobility shift and for the effect on tau's nucleation activity. We conclude that tau's activities to promote growth of existing microtubules and nucleation of new ones are differentially affected by the phosphorylation of specific tau residues. Regulation of the phosphorylation state by substrate modulation may play an important role in regulating tau's function.

Amino Acid Sequence↗

PubMed1994

Accessibility of the c-Src SH2-domain for binding is increased during mitosis.

The Src homology 2 (SH2) region is a noncatalytic domain of Src-family tyrosine kinases and other proteins which participants in inter- and intramolecular interactions of tyrosine-phosphorylated proteins. A synthetic peptide modeled on the c-Src carboxyl terminus, which contains phosphotyrosine at position 527, binds recombinant SH2 and the SH2-domain of c-Src which lacks phosphotyrosine 527. Unphosphorylated peptide does not bind detectably. Thus, the phosphorylated peptide is a specific probe for investigating SH2 accessibility. Since Src and other tyrosine kinases may participate in regulating events in mitosis, we used the SH2-binding probe to test the prediction that decreased tyrosine 527 phosphorylation would lead to increased accessibility of the c-Src SH2-domain during mitosis. Probe binding to overexpressed chicken c-Src was enhanced at least 6-fold during mitosis, indicating that the c-Src SH2-domain is more accessible in this part of the cell cycle. This suggests that there may be mitosis-specific interactions of the c-Src SH2-domain with cellular proteins in vivo.

3T3 Cells↗

PubMed1994

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

3T3 Cells↗

PubMed1994

Tyrosine dephosphorylation of pp60c-src is stimulated by a serine/threonine phosphatase inhibitor.

Incubation of NIH3T3-derived c-src overexpressor cells with okadaic acid, a specific serine/threonine phosphatase inhibitor, stimulates pp60c-src kinase activity about 2-3-fold. Activation is blocked if cells are simultaneously treated with orthovanadate, a tyrosine phosphatase inhibitor. Furthermore, okadaic acid treatment induces a small decrease in Tyr 527 phosphorylation of wild-type pp60c-src and a large decrease in Tyr 527 phosphorylation of kinase-defective pp60c-src(Lys 295-->Arg). These results suggest that the activation is mediated by okadaic acid-induced changes in tyrosine phosphorylation of pp60c-src involving 'cross-over' from serine/threonine to tyrosine signal transduction pathways. Stimulation of pp60c-src activity and Tyr 527 dephosphorylation do not require changes in serine/threonine phosphorylation of pp60c-src, suggesting that these changes result from modulation of an upstream Tyr 527 phosphatase or kinase which is itself regulated by altered serine/threonine phosphorylation. Since okadaic acid induces a pseudo-mitotic phenotype in rodent cells (K. Yamashita, H. Yasuda, J. Pines, K. Yasumoto, H. Nishitani, M. Ohtsubo, T. Hunter, T. Sugimura and T. Nishimoto, EMBO J., 9: 4331-4338, 1990), it is possible that these phenomena are induced by a biochemical mechanism similar to that which causes transient tyrosine dephosphorylation of pp60c-src during mitosis.

3T3 Cells↗

PubMed1993

Mitogenic activation of the Ras guanine nucleotide exchange factor in NIH 3T3 cells involves protein tyrosine phosphorylation.

We report biochemical evidence that epidermal growth factor and platelet-derived growth factor stimulate the Ras guanine nucleotide exchange factor activity in quiescent NIH 3T3 cells. Moreover, the exchange activity is constitutively enhanced in NIH 3T3 cells transformed by Src and ErbB2 oncogenic tyrosine protein kinases (TPKs), whereas transformation by oncogenic Mos and Raf does not alter the activity. GTPase-activating protein activity was not affected under these conditions. Overexpression of pp60c-Src mutants containing activated and suppressor TPK mutations resulted in stimulation and inhibition of the exchange factor activity, respectively. A TPK inhibitor, genistein, prevented the activation of the exchange factor in epidermal growth factor/platelet-derived growth factor-treated cells and src-transformed cells. Furthermore, the exchange factor activity bound to an anti-phosphotyrosine antibody immunoaffinity column. These findings suggest that the guanine nucleotide exchange factor, but not GTPase-activating protein, plays a major role in the Ras activation in cell proliferation initiated by growth factor receptor TPKs and malignant transformation by oncogenic TPKs and that tyrosine phosphorylation of either the exchange factor or a tightly bound protein(s) may mediate the activation of the exchange factor by these TPKs.

3T3 Cells↗

PubMed1993

Aluminum-induced nonenzymatic phospho-incorporation into human tau and other proteins.

Incubation of purified recombinant human tau protein with aluminum salts at concentrations > or = 100 microM induces aggregation of tau that prevents its entry into SDS-polyacrylamide gels and filtration through nylon membranes. This effect is noncovalent and can be reversed by addition of EDTA. However, when incubated along with ATP, GTP, or CTP, aluminum catalyzes a covalent linkage that results in incorporation of the alpha- and gamma-phosphates into the tau protein (phospho-incorporation). The sensitivity to phosphatases and partial hydrolysis and the labeling observed with ATP containing radioisotopes at different positions suggest a novel reaction in which the entire triphosphate moiety is transferred from ATP and linked to tau via an O-linkage to the alpha-phosphate. The aggregation and triphosphorylation phenomena were not catalyzed by divalent or quadrivalent cations, but similar effects were observed with some other trivalent cations. They occurred at aluminum concentrations similar to those found in human brains with Alzheimer's disease, suggesting the possibility that related reactions may have physiological significance in vivo.

Adenosine Triphosphate↗

PubMed1993

The cell cycle and c-Src.

The activity of the proto-oncogene encoded c-Src product is tightly regulated in vivo. In recent years, a model has emerged of how this regulation is achieved. In particular, protein kinases and phosphatases that are potential regulators of c-Src activity in the cell cycle have been identified and characterized.

Amino Acid Sequence↗

PubMed1993

Aluminum interaction with human brain tau protein phosphorylation by various kinases.

Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl3 at 10 nM to 10 microM range activated in-vitro [gamma-32P]ATP phosphorylation of the brain (tau) tau protein in both normal human or E. coli expressed tau forms; in the presence of the kinases P34, PKP, and PKC. However, higher concentrations of ALCl3 inhibited the tau phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl3 at 100 microM to 500 microM range induced non-enzymatic phosphorylation of tau with gamma-ATP, gamma-GTP, and alpha-GTP. AlCl3 activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of tau by Al3+ was accompanied by molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the longterm neurological effect of Al3+ in human brain leading to the formation of the neurofibrillary tangles related to Alzheimer's disease.

Adenosine Triphosphate↗

PubMed1993

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

Animals↗

PubMed1993

Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells.

Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both normal, myristoylated pp60c-src and a nonmyristoylated mutant, pp60c-src/myr-, has been used to compare the effects of preventing myristoylation on the localization of c-Src in NIH 3T3-derived overexpresser cells using immunofluorescence microscopy. During interphase, pp60c-src partitions between the plasma membrane and the centrosome, while pp60c-src/myr- is predominantly cytoplasmic but also partly nuclear. The cytoplasmic, but not the nuclear, staining can be readily washed out by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr-, in contrast with the nuclear one, does not associate tightly with structures that are insoluble in the presence of nonionic detergents. We have previously shown that during G2 phase, pp60c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that pp60c-src/myr- translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr-, but not of pp60c-src, is also observed when cells are arrested in G0 or at the G1/S transition. Furthermore, during mitosis, pp60c-src is found primarily in diffuse and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr- more specifically associates with the main components of the spindle apparatus (poles and fibers) and inside the interchromosomal space. These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts are readily moved into the nucleus. They also raise the possibility that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.

3T3 Cells↗

PubMed1993

Gap junctional communication and neoplastic transformation.

Gap junctional communication (GJC) is mediated by channels consisting of connexins and can be differentially regulated by ions, second messengers, kinases, phosphatases, and cell adhesion molecules. Tumor cells and oncogene-transformed cells often, but not always, show reduced homologous GJC between themselves. A more stringent correlation may exist between transformation and reduced heterologous communication between transformed cells and normal neighbors. Reduced GJC seems to stimulate tumor promotion but has no significant effect on the initiation phase of carcinogenesis. These effects may reflect the importance of intercellular passage of second messengers or other small molecules in cell growth control. Some evidence suggests that gap junction in combination with cell adhesion molecules can affect metastatic potential, but a clear picture has not yet emerged. Coupling and gap junction expression can be regulated both pre- and posttranslationally in oncogene-transformed cells. Src probably downregulates GJC in fibroblasts by tyrosine phosphorylation of connexin43. The Ras-induced reduction in GJC appears to be caused by decreased connexin expression. E1A, but not Myc and Fos, downregulates GJC to some extent. Artificial expression of connexin in glioma, hepatoma, chemically transformed, and src-transformed cells can restore GJC and suppress growth and/or tumorigenesis. These results argue for involvement of GJC in transformation and growth control.

Animals↗