Pp60 Src Research Articles

Differential Translocation of Phospholipase C Isozymes to Integrin-mediated Cytoskeletal Complexes in Thrombin-stimulated Human Platelets

To investigate a role of phospholipase C (PLC) isozymes in the integrin alphaIIbbeta3-mediated signaling, their location was examined in thrombin-activated human platelets, revealing different regulation of their translocation to the cytoskeleton (CSK). In resting platelets, the major PLCs such as PLCbeta2, PLCbeta3a (155 kDa), and PLCgamma2 and the minor PLCs (PLCbeta1 and PLCgamma1) were located in the Triton X-100-soluble (Tx.Sol) fraction and the membrane skeleton, whereas PLCbeta3b (140 kDa) was present only in Tx.Sol fraction when examined by Western immunoblotting. Thrombin stimulation caused a rapid and transient translocation of PLCbeta3a and PLCbeta3b and a slower accumulation of PLCbeta2 and PLCgamma2 in the reorganized CSK. The translocation to CSK of both PLCbeta3a and PLCbeta3b, but not PLCbeta2, was dependent on integrin alphaIIbbeta3-mediated aggregation. Furthermore, an actin polymerization inhibitor, cytochalasin D, or a protein tyrosine kinase inhibitor, genistein, abolished the CSK association of alphaIIbbeta3, PLCbeta3a, and PLCbeta3b. In the genistein-pretreated platelets, pp60(c-)src, Gq, and protein kinase Calpha were no longer able to associate with CSK. In contrast, these agents had no or marginal inhibitory effects on the CSK association of PLCbeta2 and Gi2. The late diacylglycerol generation induced by thrombin stimulation was significantly reduced by the genistein treatment. These results suggest that the integrin alphaIIbbeta3-mediated cytoskeletal association of PLCbeta3 is regulated by protein tyrosine kinase and also that the activation of the relocated PLC may play a role in the late platelet-to-platelet aggregation in thrombin-stimulated human platelets.

Novel phosphotyrosine mimetics in the design of peptide ligands for pp60 src SH2 domain

We have designed and synthesized a series of phosphorylated penta- and tri-peptides of general structures R x-Glu-Glu-Ile-Glu and R x-Glu- D -Trp-NH 2, where R x represents a phosphotyrosine mimetic. These peptides show binding affinity to pp60 src SH2 domain in the micromolar range. Data are presented that provide an account of their structure-activity relationships and specificity properties. We have designed and synthesized a series of phosphorylated penta- and tri-peptides of general structures R x-Glu-Glu-Ile-Glu and R x-Glu- D -Trp-NH 2, where R x represents a phosphotyrosine mimetic. These peptides show binding affinity to pp60 arc SH2 domain in the micromolar range. Data are presented that provide an account of their structure-activity relationships and specificity properties.

Distribution of myristoyl-CoA:protein N-myristoyl transferase activity in rabbit intestine

Myristoyl-CoA:protein N-myristoyl transferase (NMT) attaches the fatty acid, myristate, to the amino-terminal glycine residue of various proteins involved in cellular regulation and/or signal transduction. We report differences in the activity and properties of NMT in New Zealand rabbit small intestine, ascending colon and descending colon. The mucosa of the small intestine, ascending colon and descending colon was assayed for NMT activity using peptides of known myristoylated proteins (pp60 src and catalytic subunit of cAMP dependent protein kinase). Total NMT activity per gram tissue was 5-fold higher in the small intestine and 1.5-fold higher in the ascending colon than in the descending colon. Smooth muscle from the colon also contained low levels of NMT activity. NMT activity was 2- to 3-fold higher in the particulate fraction than in the cytosolic fraction of the mucosa in the descending colon. The apparent molecular mass of NMT in the intestine mucosa was 78 kDa.

Signalling pathways activated by endothelin stimulation of renal cells.

1. Endothelin mediates its effects in a variety of renal cells via a multiplicity of intracellular signalling pathways. 2. Stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC), resulting in the activation of inositol trisphosphate and diacylglycerol, can be detected even at picomolar concentrations of peptide. 3. Endothelin activation of cPLA2 is sensitive to ambient [Ca2+]i, is not contingent upon protein kinase C activation and is independent of PI-PLC stimulation, being coupled to the endothelin receptor in a yet to be determined manner. 4. Activation by endothelin of phosphatidylcholine-specific phospholipase D is under the dual regulation of protein kinase C and [Ca2+]i, with protein kinase C being the major regulator and [Ca2+]i playing a secondary, modulatory role. 5. Phosphatidylcholine-specific phospholipase C (PC-PLC) is stimulated by endothelin and accounts for the prolonged activation of diacylglycerol by this peptide. PC-PLC activity is critically dependent upon [Ca2+]i, whereas protein kinase C plays no role in modulating the activity of this enzyme. 6. Endothelin enhances the phosphorylation of protein tyrosine kinases, with evidence that phosphorylation of pp60 Src may be an important early event.

Solid phase synthesis of pp60 src-related phosphopeptides via ‘global’ phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2

The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60 src protein kinase family, Asn-Glu-Tyr( P)-Ser-Ala, Ala-Glu-Tyr( P)-Ser-Ala, Ala-Ser-Tyr( P)-Ser-Ala, Ala-Ser( P)-Tyr-Ser-Ala, Ala-Thr-Tyr( P)-Ser-Ala, Ala-Thr( P)-Tyr-Ser-Ala and Ala-Ser( P)-Tyr( P)-Ser-Ala, were prepared in good yield using the ‘global’ ‘phosphite-triester’ phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP® coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final ‘global’ phosphorylation of the peptide resins was accomplished using di- tert-butyl N, N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di- t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr( P) residue in the −1 position was significantly more important than the Ser( P)/Thr( P) residue in the −2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the −2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr( P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser( P), Thr( P), Glu}. In addition to the Tyr( P) residue directing phosphorylation to the + 1 position, the good phosphorylation of both ASY( P)SA and ATY( P)SA by casein kinase-2 indicated that the Tyr( P) residue was also able to direct phosphorylation to a Ser/Thr in the −1 position.

PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp 60src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.

SUPPRESSION AND RESTORATION OF V-SRC EXPRESSION IN RSV TRANSFORMED-CELLS AFTER TRANSFECTION WITH N-RAS AND ITS ANTAGONIST

Previously, it was shown that hamster cells transformed by Rous Sarcoma Virus (RSV) exhibited a decreased expression of the RSV products (including the pp60 src oncogene) when these cells were supertransfected with the N-ras oncogene. To assess the responsibility of the activated N-ras in the modulation of the RSV viral products, a strategy based on two ras antagonists was used; i.e. i) a rap1A/K-rev1 expression vector known for its capacity to revert the K-ras induced transformed phenotype and ii) a plasmid containing antisense N-ras sequence. We present data showing only the plasmid construct containing the N-ras antisense sequense could inhibit expression or N-ras and, at the same time, restore the expression of v-src, up to a level comparable to that of the parental cells. Our results support the idea that some biological switches, triggered and activated through the N-ras oncogene pathway, might modulate the promoter activity of the RSV LTR.

Selective inhibition of the tyrosine kinase pp60 src by analogs of 5,10-dihydropyrimido[4,5-b]quinolin-4(1H)-one

7,8-Dimethoxy-5,10-dihydropyrimido[4,5-b]quinolin-4(1H)-one ( 1b) has been discovered to be a potent and selective inhibitor of the tyrosine-specific kinase activity associated with pp60 c-src. Mole of inhibition studies reveal that this agent inhibits in a pure-mixed-noncompetitive mode with respect to nucleotide co-factor.

The association of pp125 FAK, pp60 Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium

The integrin α IIb β 3-mediated redistribution of the tyrosine kinases pp125 FAK and pp60 Src and the small GTP-binding proteins CDC42Hs and Rap1B from the membrane skeleton to the cytoskeleton was found to be reversible: upon prolonged platelet aggregation (up to 15 min) induced by the thrombin-receptor activating peptide (TRAP) these signalling proteins dissociated from the cytoskeleton and reappeared in the membrane skeleton. Addition of the extracellular Ca 2+ chelator EGTA and the intracellular Ca 2+ chelator BAPTA/AM 30 s after TRAP allowed platelet aggregation and the association of pp125 FAK, pp60 Src , CDC42Hs and Rap1B with the cytoskeleton, but prevented their dissociation from the cytoskeleton. The results indicate that the prolonged elevation of cytosolic Ca 2+ in stimulated platelets leads to the dissociation of signalling proteins from the cytoskeleton.

The synthesis and use of pp60 src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60 src , -Asn-Glu-Tyr 416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr( P)-containing peptide, Asn-Glu-Tyr( P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO 3Bzl 2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF 3CO 2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser( P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr( P)-residue, and (C) a Ser( P)-residue was as effective as the Tyr( P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group {i.e. Tyr(Me) and Tyr respectively} but enhanced by the introduction of the hydrophilic phosphate group {i.e. as Tyr( P)}.

Pp59 fyn and pp62 c-yes are enriched in SH-SY5Y neuroblastoma growth cones but do not associate to the 38 kDa protein which complexes with pp60 c src and pp60 c-srcN

The tyrosine-specific kinases pp60 c-src and pp60 c-srcN (pp60 src) are slightly enriched and activated in growth cones isolated from neuronally differentiating SH-SY5Y neuroblastoma cells. In the growth cones the two src isoforms are associated with a 38 kDa protein. In this report, we have compared the subcellular distribution of pp59 fyn and pp62 c-yes with that of pp60 src in differentiating SH-SY5Y cells. Like pp60 src, the other two tyrosine kinases were slightly enriched and activated in the growth cones as compared to the levels in the cell bodies. The kinase activites were 3- to 4-times higher in growth cones than in cell bodies. However, only pp60 src formed a complex with the 38 kDa protein while immunoprecipitation of pp59 fyn brought down an additional protein of 90 kDa. This may suggest that these related tyrosine kinases have different substrates and in part mediate different cellular responses in the growth cones of differentiating SH-SY5Y cells.

Histomorphometric and immunocytochemical studies of src-related osteopetrosis

“Knockout” of the src proto-oncogene in mice resulted in the unexpected development of osteopetrosis which was shown subsequently in 4–6 week old src-mutants to be due to failure of src-mutant osteoclasts to form ruffled borders. Histomorphometric analysis of vertebrae and humeri from 9–10 month old src-mutant mice has revealed that the osteopetrosis persists and that osteoclasts from these older mice also fail to form ruffled borders. Deficiency of src, however, does not impair formation of other complex cytoplasm extensions, such as respiratory cilia or intestinal villi. Immunocytochemistry using antibodies to pp60 src has shown that the src tyrosine kinase is not localized along ruffled borders of normal osteoclasts in human bone, but is expressed diffusely throughout their cytoplasm. Furthermore, pp60 src is expressed diffusely throughout the cytoplasm of non-resorbing osteoclasts in a human giant cell tumor of bone. These findings indicate that, unlike some other forms of the disorder, remission does not occur in src-deficiency-related osteopetrosis, at least by ten months of age. They also show that high expression of the pp60 src protein is not confined to actively resorbing osteoclasts, but can also occur in osteoclasts unattached to bone surfaces, and suggest that there may be a critical substrate for src that is involved in ruffled border formation.

Epinephrine induces association of pp60 src with G iα in human platelets

Using specific antibodies against the alpha subunit of the inhibitory GTP-binding protein Gi, we analyzed the association of Gi alpha with other cellular components in human platelets. Three tyrosine phosphorylated proteins with molecular mass of 63, 58, and 55 kDa were specifically associated with Gi alpha in resting platelets. Stimulation of platelets with epinephrine, but not with thrombin, induced an increase of the reactivity of the 63- and 55-kDa proteins to anti-phosphotyrosine antibodies on western blotting. By in vitro kinase assay we found that epinephrine induced the association of kinase activity with Gi alpha and that the 63-kDa protein was phosphorylated by this activity. The association of kinase activity with Gi alpha in epinephrine-stimulated platelets paralleled the association of pp60src with Gi alpha, as detected by western blotting analysis using specific anti-pp60src monoclonal antibodies. The interaction of pp60src with Gi alpha may play a role in the mechanism of platelet activation by epinephrine or in the epinephrine-induced potentiation of the action of other platelet agonists.

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60 src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60 src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accomodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.

N-myristoyl transferase assay using phosphocellulose paper binding

N-myristoyl-CoA: protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH 2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA: protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [ 3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60 src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain > spleen > heart > liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.

Powering the cell cycle

It is now over a decade since the discovery that proteins can be phosphorylated on tyrosine as well as serine and threonine residues [1]. The first of the protein-tyrosine kinases responsible for this phosphorylation to be identiffed were either oncogene products, such as pp60 src, or receptors, such as that for epidermal growth factor. The plasma membrane association of these proteins and the many tyrosine kinases subsequently isolated reinforced the view that tyrosine phosphorylation is confined to the very early events of signal transduction, such as the response to certain mitogens. However, in the past couple of years the identification of phosphotyrosine-containing proteins in yeast has shaken up the field and revealed what may be the original, fundamental application of this post-translational modification in cell-cycle control.

Cell/substratum adhesions in RSV-transformed rat fibroblasts

Cell/substratum adhesions have been studied in rat fibroblasts transformed by a ts-mutant of Rous sarcoma virus (LA-29) using light and electron microscopy and a variety of preparative methods including immunolabeling. Cells were studied both during the process of transformation i.e., shifting from 39 ° to 35 °C, and in a fully transformed state (passaged at 35 °C continuously). The typical focal contacts observed at 39 °C (restrictive temperature) were replaced by “point-contacts” (100–200 per cell) which were classified by immunolabeling as podosome-like adhesions containing actin, β 1 integrin subunit, vinculin, talin, α-actinin, and small membrane patches containing clathrin and integrin. Tyrosine-phosphorylated proteins and pp60 src were found in association with groups of small particles on the protoplasmic surface of ventral membranes by gold immunolabeling. Both types of point-contacts were visualized by electron microscopy of ultrathin sections and shadowed replicas and characterized by gold immunolabeling wherever possible. The overall composition of podosome-like adhesions is similar to focal contacts but there are differences in the three-dimensional organization of the microfilaments and in the topography of vinculin which is associated more with actin filaments than with the plasma membrane. The presence of talin and extracellular matrix receptor in podosomes together with the adhesive properties of these actin-containing structures argues against the hypothesis that pp60 src affects the interaction of actin with the plasma membrane by phosphorylating the fibronectin receptor and/or other associated proteins.

On the principles of functional ordering in biological membranes

Integrating the available data on lipid-protein interactions and ordering in lipid mixtures allows to emanate a refined model for the dynamic organization of biomembranes. An important difference to the fluid mosaic model is that a high degree of spatio-temporal order should prevail also in liquid crystalline, “fluid” membranes and membrane domains. The interactions responsible for ordering the membrane lipids and proteins are hydrophobicity, coulombic forces, Van der Waals dispersion, hydrogen bonding, hydration forces and steric elastic strain. Specific lipid-lipid and lipid-protein interactions result in a precisely controlled yet highly dynamic architecture of the membrane components, as well as in its selective modulation by the cell and its environment. Different modes of organization of the compositionally and functionally differentiated domains would correspond to different functional states of the membrane. Major regulators of membrane architecture are proposed to be membrane potential controlled by ion channels, intracellular Ca 2+, pH, changes in lipid composition due to the action of phospholipases, cell-cell coupling, as well as coupling of the membrane with the cytoskeleton and the extracellular matrix. Membrane architecture is additionally modulated due to the membrane association of ions, lip- and amphiphilic hormones, metabolites, drugs, lipid-binding peptide hormones and amphitropic proteins. Intermolecular associations in the membrane and in the membrane-cytoskeleton interface are further selectively controlled by specific phosphorylation and dephosphorylation cascades involving both proteins and lipids, and regulated by the extracellular matrix and the binding of growth factors and hormones to their specific receptor tyrosine kinases. A class of proteins coined architectins is proposed, as a notable example the pp60 src kinase. The functional role of architectins would be in causing specific changes in the cytoskeleton-membrane interface, leading to specific configurational changes both in the membrane and cytoskeleton architecture and corresponding to (a) distinct metabolic/differentiation states of the cell, and (b) the formation and maintenance of proper three dimensional membrane structures such as neurites and pseudopods.

Calpactin I in the differentiating embryonic chicken lens: mRNA levels and protein distribution

Calpactin I, one of the EDTA-extractable proteins of the lens membrane, binds phospholipid and actin in a calcium-dependent manner. It is also a known substrate of the pp60 src kinase. Analysis of embryonic chicken lens RNA with a bovine calpactin I-specific cDNA probe revealed the presence of a ∼1.8 Kb calpactin mRNA in the lens cells. Six-day embryonic chicken lenses were microdissected into central epithelium, equatorial epithelium, and fiber cells. Total cytoplasmic RNA was isolated from these samples and calpactin I mRNA levels were determined by the polymerase chain reaction (PCR) following reverse transcription (RT). Quantitative PCR indicates that the calpactin I mRNA levels in the equatorial epithelium are greater than in the central epithelium by a factor of 12.7 ± 2.7. Calpactin I mRNA in fiber cells is an additional 3.5 ± 1.5 times greater than in the equatorial epithelium. Whole mounts of embryonic chicken lens epithelia and histological sections of whole lenses were also examined with an antibody directed against chicken calpactin I. Calpactin I was predominantly localized in a punctate distribution in equatorial epithelial cells and near the plasma membrane of elongate fiber cells. The elevated levels of calpactin I mRNA observed in the equatorial epithelium and fiber cells and the immunological localization of the protein suggest a possible role of calpactin I in the elongation of fiber cells during lens differentiation.

Myristoylated [formula omitted]-oncogene products are potently detected by the monoclonal antibody to the NH 2-terminal myristoyl-Gly-Ser-Ser-Lys [formula omitted]-peptide

Monoclonal antibodies were raised against a synthetic NH 2-terminal myristoyl tetrapeptide ( N -myristoyl-Gly-Ser-Ser-Lys) which is characteristic of an NH 2-terminal portion of pp60 src , the transforming protein of src -oncogene. The antibody reacted with the albumin conjugated with both the N -myristoyl and N -lauroyl-tetrapeptides, but concentrations at which 50% of the immunoreaction was inhibited were 5 pmol for the N -myristoyl and 830 pmol for N -lauroyl tetrapeptidyl albumin. On the other hand, N -palmitoyl tetrapeptidyl and underivatized albumin, and Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys octapeptide had no effects. These results suggest a high affinity of the antibody for an N -myristoyl-Gly-Ser-Ser-Lys moiety. src -Oncogene products in Rous sarcoma virus-transformed cells and human colon carcinoma tumor cells were selectively identified as myristoylated pp60 src by immunoprecipitation analyses with the antibody.