• A myristoyl/phosphotyrosine switch regulates c-Abl.
  Hantschel O., Nagar B., Guettler S., Kretzschmar J., Dorey K., Kuriyan J., Superti-Furga G. (2003). Cell 112(6), 845-857
  PubMed

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The c-Abl tyrosine kinase is inhibited by mechanisms that are poorly understood. Disruption of these mechanisms in the BCR-Abl oncoprotein leads to several forms of human leukemia. We found that, like Src kinases, c-Abl 1b is activated by phosphotyrosine ligands. Ligand-activated c-Abl is particularly sensitive to the anti-cancer drug STI-571/Gleevec™/imatinib (STI-571). The SH2 domain-phosphorylated tail interaction in Src kinases is functionally replaced in c-Abl by an intramolecular engagement of the N-terminal myristoyl modification with the kinase domain. Functional studies coupled with structural analysis (Nagar et al., accompanying paper) define a myristoyl/phosphotyrosine switch in c-Abl which regulates docking and accessibility of the SH2 domain. This mechanism offers an explanation for the observed cellular activation of c-Abl by tyrosine-phosphorylated proteins, the intracellular mobility of c-Abl and provides further insights into the mechanism of action of STI-571.

• Structural basis for the autoinhibition of c-Abl tyrosine kinase.
  Nagar B., Hantschel O., Young M., Scheffzek K., Veach D., Bornmann W., Clarkson B., Superti-Furga G., Kuriyan J. (2003). Cell 112(6), 859-871
  PubMed

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view PDB coordinates (1OPJ; 1OPL; 1OPK)

c-Abl is normally regulated by an auto-inhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the auto-inhibited form of the closely related Src kinases, by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Auto-inhibited c-Abl forms an assembly which is strikingly similar to that of inactive Src kinases, but with specific differences that explain the differential ability of the drug Imatinib to inhibit the catalytic activity of Abl but not that of c-Src.

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• c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis.
  Furstoss, O., Dorey, K., Simon, V., Barila, D., Superti-Furga, G., Roche, S. (2002). EMBO J 21(4),514-24
  PubMed

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.

• Autoinhibition of c-Abl.
  Pluk H., Dorey K., Superti-Furga G. (2002). Cell 108(2),247-59
  PubMed

Despite years of investigation, the molecular mechanism responsible for regulation of the c-Abl tyrosine kinase has remained elusive. We now report inhibition of the catalytic activity of purified c-Abl in vitro, demonstrating that regulation is an intrinsic property of the molecule. We show that the interaction of the N-terminal 80 residues with the rest of the protein mediates autoregulation. This N-terminal "cap" is required to achieve and maintain inhibition, and its loss turns c-Abl into an oncogenic protein and contributes to deregulation of BCR-Abl.

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• Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase.
  Dorey K., Engen J.R., Kretzschmar J., Wilm M., Neubauer G., Schindler T., Superti-Furga G. (2001). Oncogene 20(56),8075-84
  PubMed

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.

• Dynamic coupling between the SH2 and SH3 domains of c -Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.
  Young M.A., Gonfloni S., Superti-Furga G., Roux B., Kuriyan J. (2001). Cell 105(1),115-26
  PubMed

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.

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• Nuclear tyrosine phosphorylation: the beginning of a map.
  Cans C., Mangano R., Barila D., Neubauer G., Superti-Furga G. (2000). Biochem Pharmacol 60(8),1203-15
  PubMed

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.

• Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src.
  Gonfloni, S., Weijland A., Kretzschmar, J., and Superti-Furga, G. (2000). Nat. Stuct. Biol. 7(4), 281-286.
  PubMed

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 C-helix in the catalytic domain is positioned such that the formation of the Glu 310–Lys 295 salt bridge isprecluded, 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 C-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the C-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.

• A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK.
  Barilá, D., Mangano, R., Gonfloni, S., Kretzschmar, J., Moro, M., Bohmann, D. and Superti-Furga, G. (2000). EMBO J. 19(2), 273-281.
  PubMed

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 phosphorylates 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.

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• Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling.
  Frischknecht F, Moreau V, Rottger S, Gonfloni S, Reckmann I, Superti-Furga G, Way M. (1999). Nature 401, 926-9.
  PubMed   Press release

Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the eventsoccurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, usesactin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknownphosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R bySrc-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor proteinNck 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 theactin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actinpolymerization at the plasma membrane.

• Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone- induced G2/M transition by oncogenic forms.
  Dorey, K., Barilá, D., Gavin, A.C., Nebreda, A.R. and Superti-Furga, G. (1999). Biol. Chem. 380, 223-230.

• Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.
  Mason, C.S., Springer, C.J., Cooper, R.G., Superti-Furga, G. , Marshall, C.J. and Marais, R. (1999). EMBO J. 18, 2137-2148.
   

• Leucine 255 of src couples intramolecular interactions to inhibition of catalysis.
  Gonfloni, S., Frischknecht, F., Way, M. & Superti-Furga, G. (1999). Nat. Struct. Biol. Aug;6(8), 760-4.
  PubMed.   Press release

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.

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• An intramolecular SH3-domain interaction regulates c-Abl activity.
  Barilá, D. & Superti-Furga, G. (1998). Nat. Genet. Mar 18(3), 280-282.
  PubMed

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.

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• Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.
  Migliaccio, E., Mele, S., Salcini, A.E., Pelicci, G., Venus Lai, K.-M., Superti-Furga, G., Pawson, T., Di Fiore, P.P., Lanfrancone, L. and Pelicci, P.G. (1997).  EMBO J. 16, 706-716.

• The cleavage stage (CS) histones of the sea urchin are encoded by a replacement histone gene family expressed during oogenesis and early development: functional equivalence of the CSH1 and frog H1m proteins.
  Mandl, B., Brandt, W.F., Superti-Furga, G., Graninger, P.G., Birnstiel, M.L. and Busslinger, M. (1997). Mol. Cell. Biol. 17, 1189-1200.
   

• Src regulated by C-terminal phosphorylation is monomeric.
  Weijland, A., Williams, J. C., Neubauer, G., Courtneidge, S.A., Wierenga, R.K. and Superti-Furga, G. (1997).  Proc. Natl. Acad. Sci. USA 94, 3590-3595.

• A crystal milestone: the structure of regulated Src.
  Superti-Furga, G. & Gonfloni, S. (1997).   Bioessays Jun 19(6), 447-450.
  PubMed

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.

 

• Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?
  Baldin, V., Cans, C., Superti-Furga, G., Ducommun, B. (1997). Oncogene 14, 2485-2495.

• A functional screen for regulators of the c-Abl protein tyrosine kinase.
  Moro, M., Walkenhorst, J., Goga, A., Witte, O.N., and Superti-Furga, G. (1997).  Leukemia 11, Suppl. 3, 313-315.

• The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.
  Williams, J.C., Weijland, A., Gonfloni, S., Thompson, A., Courtneidge, S.A., Superti-Furga, G. & Wierenga, R.K. (1997).  J. Mol. Biol. Dec 19 274(5), 757-775.
  PubMed

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. Copyright 1997 Academic Press.

 

• The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.
  Gonfloni, S., Williams, J.C., Hattula, K., Weijland, A., Wierenga, R.K. & Superti-Furga, G. (1997).  EMBO J. Dec 15 16(24), 7261-7271.
  PubMed

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.

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• Analysis of human c-Abl tyrosine kinase activity and regulation in S. pombe.
  Walkenhorst, J., Goga, A., Witte, O.N. & Superti-Furga, G. (1996). Oncogene Apr 4 12(7), 1513-1520.
  PubMed

c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells. Several mutations, including deletions of the SH3 domain, can activate abl and convert it into an oncogene. To study c- Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion. c-Abl, but not a kinase inactive form of the molecule, causes growth arrest followed by death of the cells. Concomitant to Abl expression we observed extensive phosphorylation of endogenous proteins on tyrosine. Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme. An SH3 domain deletion mutant of Abl is equally active as wild type c-Abl in yeast, even under conditions allowing detection of subtle differences. These results demonstrate that there is no intrinsic regulation of c-Abl kinase activity via the SH3 domain and suggest that the inhibitory effect of the SH3 domain observed in mammalian cells is medicated by a factor that is absent in fission yeast. Expression of Ab1 S.pombe provides a novel quantitative assay for ab1 activity and regulation.

 

• A functional screen in yeast for regulators and antagonizers of heterologous protein tyrosine kinases.
  Superti-Furga, G., Jonsson, K. & Courtneidge, S.A. (1996). Nat. Biotechnol. May 14(5), 600-605.
  PubMed

Tyrosine phosphorylation exerts a pivotal role in cell regulation processes of higher eukaryotes. Tight control of the activity of protein tyrosine kinases is crucial for ordered phosphorylation to occur. We have developed a functional screen for tyrosine kinase regulators using c-Src, the first cellular protein tyrosine kinase described, as a prototype; and fission yeast, Schizosaccharomyces pombe, as a genetically amenable host system. Inducible expression of c- Src in fission yeast is lethal. We have screened human cDNA libraries for clones able to counteract the lethal effect of Src. Two different classes of cDNAs, which we called SAS for sequences antagonizing Src, were obtained. The first class encodes for the protein tyrosine kinase Csk, known to regulate Src activity through phosphorylation of the C- terminal tyrosine. The second class consists of clones encoding three different tyrosine phosphatases, counteracting Src action by dephosphorylation of Src substrates and by dephosphorylation of Src itself. The system described here can be applied to identify regulators of other heterologous tyrosine kinases, including receptor-type tyrosine kinases, which impair growth of S. pombe. 

 

• Growth controllers knocked out.
  Superti-Furga, G. (1996).  (Meeting report). Trends Genet. 12, 324-325.

• Purification and characterization of the catalytic domain of Src expressed in S.pombe: comparison of unphosphorylated and tyrosine phosphorylated species.
  Weijland, A., Neubauer, G., Courtneidge, S.A., Mann, M., Wierenga, R. and Superti-Furga, G. (1996).  Eur. J. Biochem. 240, 756-764.
   

• Fission yeast pmk1+ gene encodes a novel MAPK homologue which regulates cell integrity and functions coordinately with the PKC pathways.
  Toda, T, Dhut, S., Superti-Furga, G., Gotoh, Y., Nishida, E. Sugiura, R. and Kuno, T. (1996). Mol. Cell. Biol. 16, 6752-6764.
   

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• Structure-function relationships in Src family and related protein kinases.
  Superti-Furga, G. and Courtneidge, S.A. (1995).  Bioessays 17, 321-330. (Review)

• Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra-and intermolecular interactions.
  Erpel, T., Superti-Furga,G. and Courtneidge, S.A. (1995).  EMBO J. 14, 963-975.

• Regulation of the Src protein tyrosine kinase.
  Superti-Furga, G. (1995).  FEBS Letters. 369, 62-66. (Review)

• Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk.
  Koegl, M., Courtneidge, S.A. and Superti-Furga, G. (1995). Oncogene 11, 2317-2329.

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• Csk inhibition of Src activity requires both the SH2 and SH3 domains of Src.
  Superti-Furga, G., Fumagalli, S., Koegl, M., Courtneidge, S.A., and Draetta, G. (1993) . EMBO J. 12, 2625-2634.