Biomedical subjects

G Superti-Furga

Publications and source records attributed to G Superti-Furga.

At least 19 recordsLinked to original sources

PubMed2001

Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase.

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.

Blotting, Western↗

PubMed2001

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

Amino Acid Substitution↗

PubMed2000

Nuclear tyrosine phosphorylation: the beginning of a map.

Tyrosine phosphorylation is usually associated with cytoplasmic events. Yet, over the years, many reports have accumulated on tyrosine phosphorylation of individual molecules in the nucleus, and several tyrosine kinases and phosphatases have been found to be at least partially nuclear. The question arises as to whether nuclear tyrosine phosphorylation represents a collection of loose ends of events originating in the cytoplasm or if there may be intranuclear signaling circuits relying on tyrosine phosphorylation to regulate specific processes. The recent discovery of a mechanism causing nuclear tyrosine phosphorylation has prompted us to review the cumulative evidence for nuclear tyrosine phosphorylation pathways and their possible role. While we found that no complex nuclear function has yet been shown to rely upon intranuclear tyrosine phosphorylation in an unambiguous fashion, we found a very high number of compelling observations on individual molecules that suggest underlying networks linking individual events. A systematic proteomics approach to nuclear tyrosine phosphorylation should help chart possible interaction pathways.

Active Transport, Cell Nucleus↗

PubMed2000

Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway.

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.

Animals↗

PubMed2000

A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK.

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.

Amino Acid Sequence↗

PubMed2000

Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src.

The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the alphaC-helix with the activation loop is a central component of this regulatory system. Our data suggest a bidirectional regulation mechanism in which the regulatory domains inhibit Src activity, and Src activity controls the availability of the regulatory domains. By this mechanism, Src family kinases can be activated by proteins phosphorylating or changing the conformation of the catalytic domain. Once active, Src family kinases become less prone to regulation, implying a positive feedback loop on their activity.

Amino Acid Sequence↗

PubMed1999

Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.

Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the events occurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, uses actin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknown phosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck and the recruitment of the Ena/VASP family member N-WASP to the site of actin assembly. We also show that Nck and N-WASP are essential for the actin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actin polymerization at the plasma membrane.

Actins↗

PubMed1999

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.

Animals↗

PubMed1999

Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis.

The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255-pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.

Catalysis↗

PubMed1999

Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone-induced G2/M transition by oncogenic forms.

Deregulated activity of the Abl protein tyrosine kinase is oncogenic in humans and in animals. The normal cellular form of the enzyme is maintained at a low state of activity by mechanisms that have not yet been entirely elucidated. In particular, little is known about the trans-acting cellular factors involved. We have tested the activity of human c-Abl microinjected into oocytes of Xenopus laevis. In contrast to versions of Abl capable of transforming mammalian cells, which were highly active when introduced into oocytes, the activity of wild type c-Abl was inhibited. Oncogenic forms of Abl efficiently enhanced the ability of Xenopus oocytes to enter M phase following stimulation by progesterone. Abl-enhanced maturation was normal as judged by accumulation of Mos as well as activation of MAP kinase and Cdc2/CyclinB (MPF). Concomitant with maturation and activation of these kinases, Abl became extensively phosphorylated. Altogether, this suggests that an SH3 domain-dependent Abl regulation mechanism similar to the one observed in mammalian cells operates in Xenopus oocytes. Maturation enhancement by microinjection into Xenopus oocytes represents a useful novel assay for analyzing Abl activity. Moreover, the Xenopus oocyte may be a convenient source of trans-acting Abl regulators for biochemical studies.

Animals↗

PubMed1998

An intramolecular SH3-domain interaction regulates c-Abl activity.

The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase (c-Abl) that has been implicated in processes of cell differentiation, cell division, cell adhesion and stress response. Alterations of ABL1 by chromosomal rearrangement or viral transduction can lead to malignant transformation. Activity of the c-Abl protein is negatively regulated by its SH3 domain through an unknown mechanism, and deletion of the SH3 domain turns ABL1 into an oncogene. We present evidence for an intramolecular inhibitory interaction of the SH3 domain with the catalytic domain and with the linker between the SH2 and catalytic domain (SH2-CD linker). Site-directed mutations in each of these three elements activate c-Abl. Mutations in the linker cause a conformational change of the molecule and increase binding of the SH3 domain to peptide ligands. Individual mutation of two charged residues in the SH3 and catalytic domain activates c-Abl, while inhibition is restored in the double reciprocal mutant. We propose that regulators of c-Abl will have opposite effects on its activity depending on their ability to favour or disrupt these intramolecular interactions.

3T3 Cells↗

PubMed1997

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.

Amino Acid Sequence↗

PubMed1997

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.

3T3 Cells↗

PubMed1997

Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?

CDC25B2, a protein tyrosine phosphatase closely related to the putative CDC25B oncogene, was identified in a Burkitt lymphoma cDNA library. CDC25B2 differs from CDC25B by a 14 residue insertion and a 41 residue deletion, which are both located in the amino-terminal region of the protein, upstream of the catalytic domain. Examination of the genomic sequence revealed that CDC25B1 (formerly B) and CDC25B2 are splice variants of the same gene. A third variant, CDC25B3, that carries both the 14 and the 41 residue sequences was also identified in the same cDNA library. All three variants were detected in a panel of human primary culture and cell lines, although at different levels. In primary fibroblasts and in HeLa cells the CDC25B expression is cell cycle regulated, reaching a maximum in G2-phase. In vitro, CDC25B1 phosphatase is slightly more active than CDC25B2 and B3. However, episomal overexpression of the three CDC25B variants in fission yeast reveals that in vivo, CDC25B2 is largely more active than either B1 or B3 (B2>B3>B1) both to complement a thermosensitive S pombe CDC25 activity and to act as a mitotic inducer. Alternative splicing of CDC25B may therefore contribute to the control of cell proliferation.

Alternative Splicing↗

PubMed1997

Src regulated by C-terminal phosphorylation is monomeric.

The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure of the enzyme or if it occurred within a monomeric form of Src. For this purpose we purified to homogeneity a chicken Src form lacking the unique domain from Schizosaccharomyces pombe cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate Tyr-527 efficiently. Mass spectrometric analysis showed that purified Src was homogeneously phosphorylated at Tyr-527. The enzyme was in the regulated form, because it could be activated by a phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 A with space group P2(1)2(1)2(1) and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.

Amino Acid Sequence↗

PubMed1997

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

3T3 Cells↗

PubMed1997

A crystal milestone: the structure of regulated Src.

The viral and cellular forms of the Src protein tyrosine kinases take a prototypic role in oncology and signal transduction research, by virtue of being holders of an impressive number of 'firsts'. Our understanding of the biochemistry and physiology of Src has therefore always been used as a reference for our general advancement in the field of protein phosphorylation and growth control. The recent solution of the crystal structure of two members of the Src family represents a milestone in these disciplines and, as usual, provides a general lookout post for developments to come.

Animals↗

PubMed1997

The five cleavage-stage (CS) histones of the sea urchin are encoded by a maternally expressed family of replacement histone genes: functional equivalence of the CS H1 and frog H1M (B4) proteins.

The cleavage-stage (CS) histones of the sea urchin are known to be maternally expressed in the egg, have been implicated in chromatin remodeling of the male pronucleus following fertilization, and are the only histone variants present in embryonic chromatin up to the four-cell stage. With the help of partial peptide sequence information, we have isolated and identified CS H1, H2A, H2B, H3, and H4 cDNAs from egg poly(A)+ mRNA of the sea urchin Psammechinus miliaris. All five CS proteins correspond to replacement histone variants which are encoded by replication-independent genes containing introns, poly(A) addition signals, and long nontranslated sequences. Transcripts of the CS histone genes could be detected only during oogenesis and in development up to the early blastula stage. The CS proteins, with the exception of H4, are unique histones which are distantly related in sequence to the early, late, and sperm histone subtypes of the sea urchin. In contrast, the CS H1 protein displays highest sequence homology with the H1M (B4) histone of Xenopus laevis. Both H1 proteins are replacement histone variants with very similar developmental expression profiles in their respective species, thus indicating that the frog H1M (B4) gene is a vertebrate homolog of the CS H1 gene. These data furthermore suggest that the CS histones are of ancient evolutionary origin and may perform similar conserved functions during oogenesis and early development in different species.

Amino Acid Sequence↗