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More Cables to Abl
Neuron, Vol. 26, 543–550, June, 2000, Copyright 2000 by Cell Press
Previews
More Cables to Abl Life is complex. Take for example the abelson gene, named for the individual who discovered a transforming retrovirus capable of turning normal cells into cancer cells. abl was subsequently cloned from both the virus and from normal cells and found to encode a cytoplasmic protein tyrosine kinase (PTK) that, in the oncogenic state, is essentially a constitutively active enzyme. Normal Abl function is very important for humans. The most glaring example of this is in chronic myelogenous leukemia (CML). Almost invariably, all people who suffer from this cancer show the hallmark Philadelphia chromosome translocation which involves the abl gene. After decades of work by many scientists, great progress has been made at identifying roles for Abl in the cell. Yet, despite the many advances, there remains no comprehensive model for Abl function. In addition to its PTK catalytic domain, which phosphorylates substrates on tyrosine residues, Abl has a number of protein interaction domains including Src homology domains 2 and 3 (SH2 and SH3), nuclear localization signals, and DNA and actin binding domains. Abl is expressed in many cell types, is found in a number of cellular compartments, can physically associate with a broad range of targets, and appears to function in a diverse array of signaling pathways. In the latest act of this molecular drama, Tsai and colleagues in this issue of Neuron (Zukerberg et al., 2000) add another fascinating piece to the story by discovering a novel protein, termed Cables, that links Abl to cyclin-dependent kinase 5 (Cdk5). Cdk5 has been a bit of a mystery in its own right. Originally discovered by virtue of its biochemical and sequence similarities to Cdc2, it was thought to potentially be important in the control of the cell cycle (Lew et al., 1992; Meyerson et al., 1992). In fact, Cdk5 appears to be active only in differentiated neurons, where it associates with a neural-specific regulatory subunit, termed p35 (Lew et al., 1994; Tsai et al., 1994). Evidence suggests that the Cdk5/p35 serine/threonine kinase plays an important role in regulating N-cadherin-mediated cell adhesion, neuronal migration, and neurite outgrowth, perhaps by its ability to phosphorylate and inhibit the Pak1 kinase. Like Abl, Cdk5 has possible connections to the cytoskeleton and human pathology. For instance, recent data suggest that -amyloid peptide can activate Cdk5 kinase, which ultimately leads to the hyperphosphorylation of Tau (Lee et al., 2000). Hyperphosphorylated Tau is then unable to bind microtubules and aggregates into paired helical filaments, components of the neurofibrillary tangles that deposit in the brains of people suffering from Alzheimer’s disease and other neurodegenerative disorders. To discover additional substrates for Cdk5, Zukerberg et al. (2000) undertook a yeast two-hybrid screen using a kinase inactive form of the protein as bait and identified Cables, an interacting protein of 568 amino acids that
contains an area of weak homology to cyclin A and cyclin C. The authors went on to show that Cables bound to Cdk5 and that it could function as a substrate for phosphorylation by the Cdk5/p35 kinase. Interestingly, there appears to be some form of competition for binding to Cdk5 in that Cdk5/Cables and Cdk5/p35 protein complexes can be detected but Cables/Cdk5/p35 trimolecular complexes cannot. In addition to the C-terminal Cdk5 binding domain, Cables has six potential SH3 binding motifs (PXXP) clustered around its N terminus, two of which are similar to motifs known to bind the Abl SH3 domain. Biochemical tests determined that Cables does bind to Abl and that a trimolecular complex of Cdk5, Abl, and Cables exists in vivo. These results provide a critical link between Abl and Cdk5. The merging of Cdk5 and Abl pathways opens a door full of potential for speculation and future experimentation. For instance, how does the connection between Cdk5, Cables, and Abl fit into what we already know about the normal biological functions of Abl? Some of the earliest insights into the normal functions of Abl were obtained in studies with Drosophila that began in Mike Hoffmann’s laboratory in the 1980s. Indeed, the first “cable” to Abl is a gene called disabled, whose existence was pointed out 13 years ago as being a locus that, in the heterozygous mutant state, exacerbates the Abl mutant phenotypes (Henkemeyer et al., 1987). By characterizing such dosage-sensitive genes which can modify the Abl mutant phenotypes, a number of key proteins that function in the Abl pathway have since been identified. In addition to Disabled (a PTB domain–containing Abl substrate), mutations in Enabled (a proline-rich, tyrosine phosphorylated protein that binds Abl, Profilin, and actin), Dlar (a transmembrane tyrosine phosphatase), Trio (a Rac guanine nucleotide exchange factor), Chickadee (Profilin), Armadillo (-catenin), and Notch (a transmembrane receptor) have all been shown to modify the Abl mutant phenotypes (Lanier and Gertler, 2000). Further expanding the realm of Abl, Disabled and Enabled have themselves been implicated in important functions in flies and mammals, including the Reelin pathway, which is important for the normal layering of cortical neurons (Cooper and Howell, 1999). Many of the pathways these molecules function in are also implicated in Cdk5/p35 signaling, most notably in cadherin–catenin signaling and in cortical layering (see below). Thus, even without this new molecular connection between Abl and Cdk5, the two were already poised to converge. What is the purpose of Cables’ ability to bridge Cdk5 with Abl? All three proteins colocalize within cortical axons, particularly in their growth cones. One interesting possibility is that Cables and Abl both function as adaptor or scaffolding proteins to bind to Cdk5 and control its subcellular location in the neuron. Evidence for a noncatalytic adaptor-like function for Abl can be found in Drosophila, where it was demonstrated that the kinase activity of Abl was not needed to rescue the mutant phenotypes (Henkemeyer et al., 1990). Instead, it was found that two noncatalytic domains, one N terminal and the other C terminal, were absolutely essential for
Neuron 544
normal Abl function and that this function was somehow related to the ability of the Abl protein to properly localize within the axons of the embryonic CNS. Interestingly, it appears that in addition to the Abl SH3 domain, Cables also binds to two other regions of the Abl protein, one of which is the C-terminal domain and the other undefined (presumably the N terminus or the kinase domain). It may, therefore, be possible that the trimolecular interaction between Abl, Cables, and Cdk5 is required for the proper localization of the complex within the cell (namely the cytoskeleton), which could lead to the efficient phosphorylation of the Abl and Cdk5/p35 substrates that affect cytoskeletal dynamics. It will be interesting to determine whether Abl tyrosine kinase activity is essential for Cdk5/p35 function and what affect N-terminal and C-terminal domain mutations of Abl might have on Cdk5 localization. A search of the Drosophila genome indicates that a Cables-related sequence is indeed present (GenBank accession number AAF58349). This will permit genetic studies in the fly to investigate if wild-type Abl protein is important for Cables and Cdk5 localization and whether mutations in Cables or Cdk5 modify the Abl mutant phenotypes (and vice versa). The idea that Abl in mammals might have an essential function in Cdk5 signaling that is independent of its tyrosine kinase activity and more consistent with it acting as an adaptor is supported by mouse knockout studies that showed a truncated Abl protein that retains its kinase activity fails to function in the mouse, just like the Drosophila studies showed (Schwartzberg et al., 1991). However, it is important to point out that Abl can phosphorylate Cdk5 on tyrosine residue 15, and this increases the catalytic activity of the Cdk5 kinase domain. To further complicate the matter, Cdk5 is only active when bound to p35, and presumably the Abl/Cables/Cdk5 complex excludes p35 from binding. Thus, perhaps Abl and Cables are both adaptors that help colocalize Cdk5 to the proper subcellular compartment, and the Abl catalytic domain functions, in a regulated fashion, to activate Cdk5 serine/threonine kinase activity by phosphorylation of residue 15. But at this step, Cdk5 would then have to leave the Abl/Cables complex and bind p35 such that it can then phosphorylate its own target proteins, like Pak1, Tau, or -catenin. Unfortunately, the mechanisms that lead to the activation of the Abl tyrosine kinase domain in vivo are still somewhat of a mystery (Plattner et al., 1999). Beyond potentially elucidating Abl’s role as an adaptor protein, the new connection between Cables and Cdk5 reinforces the idea that Abl has important roles in regulating the cytoskeleton. Several lines of evidence implicate Abl as a key regulator of axonogenesis and cortical neuron migration. For example, Abl may lie downstream of a signaling cascade involving Reelin, very low density lipoprotein receptors, and murine Disabled (Scrambler), which have all been shown to be essential in the formation of the cortical neuron layers (Cooper and Howell, 1999). Quite interestingly, Cdk5 and p35 mutant mice also show cortical layering defects, suggesting that Abl, Cables, and Cdk5/p35 may all play a role in some aspect of Reelin signaling. To begin to uncover potential roles for Cables in cytoskeletal events, Zukerberg et al. (2000) established a neurite outgrowth assay using cultured cortical neurons. They found that
ectopic Cables expression inhibited neurite outgrowth, while ectopic Abl had the opposite effect and resulted in the elaboration of longer neurites. Although it is unclear exactly what these in vitro assays mean for the function of these molecules in vivo during brain development, they do provide a system to evaluate potentially important biological activities. For instance, it might be possible to elucidate the roles of Trio, Enabled, and Profilin with similar in vitro assays, particularly as they relate to Abl and Cables and ultimately in their potential regulation of actin and cytoskeletal dynamics at the growth cone. If we stop and try to connect the dots, it is clear that Abl and its partners have multiple roles in neuronal migration and axonal patterning, with a sum that points to Abl’s most physiologically relevant function being the regulation of cytoskeletal events. This new connection of Abl, Cables, and Cdk5 adds yet another potential mechanism by which diverse signals are integrated within the cell to control its architecture. The merging of Abl and Cdk5 is the latest in the ongoing search for an understanding of the signaling networks and protein– protein interactions that govern cytoskeletal dynamics. Clearly, this story is not over and much more research will be required to elucidate the biology of this intriguing protein in attempts to understand why all these “cables” lead to Abl. Chad A. Cowan and Mark Henkemeyer Center for Developmental Biology University of Texas Southwestern Medical Center Dallas, Texas 75235 Selected Reading Cooper, J.A., and Howell, B.W. (1999). Cell 97, 671–674. Henkemeyer, M.J., Gertler, F.B., Goodman, W., and Hoffmann, F.M. (1987). Cell 51, 821–828. Henkemeyer, M., West, S.R., Gertler, F.B., and Hoffmann, F.M. (1990). Cell 63, 949–960. Lanier, L.M., and Gertler, F.B. (2000). Curr. Opin. Neurobiol. 10, 80–87. Lee, M.-S., Kwon, Y.T., Li, M., Peng, J., Friedlander, R.M., and Tsai, L.-H. (2000). Nature 405, 360–364. Lew, J., Beaudette, K., Litwin, C.M., and Wang, J.H. (1992). J. Biol. Chem. 267, 13383–13390. Lew, J., Huang, Q.Q., Qi, Z., Winkfein, R.J., Aebersold, R., Hunt, T., and Wang, J.H. (1994). Nature 371, 423–426. Meyerson, M., Enders, G.H., Wu, C.L., Su, L.K., Gorka, C., Nelson, C., Harlow, E., and Tsai, L.-H. (1992). EMBO J. 11, 2909–2917. Plattner, R., Kadlec, L., DeMali, K.A., Kazlauskas, A., and Pendergast, A.M. (1999). Genes Dev. 13, 2400–2411. Schwartzberg, P.L., Stall, A.M., Hardin, J.D., Bowdish, K.S., Humaran, T., Boast, S., Harbison, M.L., Robertson, E.J., and Goff, S.P. (1991). Cell 65, 1165–1175. Tsai, L.-H., Delalle, I., Caviness, V.S., Chae, T., and Harlow, E. (1994). Nature 371, 419–423. Zukerberg, L.R., Patrick, G.N., Nikolic, M., Humbert, S., Wu, C.-L., Lanier, L.M., Gertler, F.B., Vidal, M., Van Etten, R.A., and Tsai, L.-H. (2000). Neuron 26, this issue, 633–646.
Previews
More Cables to Abl Life is complex. Take for example the abelson gene, named for the individual who discovered a transforming retrovirus capable of turning normal cells into cancer cells. abl was subsequently cloned from both the virus and from normal cells and found to encode a cytoplasmic protein tyrosine kinase (PTK) that, in the oncogenic state, is essentially a constitutively active enzyme. Normal Abl function is very important for humans. The most glaring example of this is in chronic myelogenous leukemia (CML). Almost invariably, all people who suffer from this cancer show the hallmark Philadelphia chromosome translocation which involves the abl gene. After decades of work by many scientists, great progress has been made at identifying roles for Abl in the cell. Yet, despite the many advances, there remains no comprehensive model for Abl function. In addition to its PTK catalytic domain, which phosphorylates substrates on tyrosine residues, Abl has a number of protein interaction domains including Src homology domains 2 and 3 (SH2 and SH3), nuclear localization signals, and DNA and actin binding domains. Abl is expressed in many cell types, is found in a number of cellular compartments, can physically associate with a broad range of targets, and appears to function in a diverse array of signaling pathways. In the latest act of this molecular drama, Tsai and colleagues in this issue of Neuron (Zukerberg et al., 2000) add another fascinating piece to the story by discovering a novel protein, termed Cables, that links Abl to cyclin-dependent kinase 5 (Cdk5). Cdk5 has been a bit of a mystery in its own right. Originally discovered by virtue of its biochemical and sequence similarities to Cdc2, it was thought to potentially be important in the control of the cell cycle (Lew et al., 1992; Meyerson et al., 1992). In fact, Cdk5 appears to be active only in differentiated neurons, where it associates with a neural-specific regulatory subunit, termed p35 (Lew et al., 1994; Tsai et al., 1994). Evidence suggests that the Cdk5/p35 serine/threonine kinase plays an important role in regulating N-cadherin-mediated cell adhesion, neuronal migration, and neurite outgrowth, perhaps by its ability to phosphorylate and inhibit the Pak1 kinase. Like Abl, Cdk5 has possible connections to the cytoskeleton and human pathology. For instance, recent data suggest that -amyloid peptide can activate Cdk5 kinase, which ultimately leads to the hyperphosphorylation of Tau (Lee et al., 2000). Hyperphosphorylated Tau is then unable to bind microtubules and aggregates into paired helical filaments, components of the neurofibrillary tangles that deposit in the brains of people suffering from Alzheimer’s disease and other neurodegenerative disorders. To discover additional substrates for Cdk5, Zukerberg et al. (2000) undertook a yeast two-hybrid screen using a kinase inactive form of the protein as bait and identified Cables, an interacting protein of 568 amino acids that
contains an area of weak homology to cyclin A and cyclin C. The authors went on to show that Cables bound to Cdk5 and that it could function as a substrate for phosphorylation by the Cdk5/p35 kinase. Interestingly, there appears to be some form of competition for binding to Cdk5 in that Cdk5/Cables and Cdk5/p35 protein complexes can be detected but Cables/Cdk5/p35 trimolecular complexes cannot. In addition to the C-terminal Cdk5 binding domain, Cables has six potential SH3 binding motifs (PXXP) clustered around its N terminus, two of which are similar to motifs known to bind the Abl SH3 domain. Biochemical tests determined that Cables does bind to Abl and that a trimolecular complex of Cdk5, Abl, and Cables exists in vivo. These results provide a critical link between Abl and Cdk5. The merging of Cdk5 and Abl pathways opens a door full of potential for speculation and future experimentation. For instance, how does the connection between Cdk5, Cables, and Abl fit into what we already know about the normal biological functions of Abl? Some of the earliest insights into the normal functions of Abl were obtained in studies with Drosophila that began in Mike Hoffmann’s laboratory in the 1980s. Indeed, the first “cable” to Abl is a gene called disabled, whose existence was pointed out 13 years ago as being a locus that, in the heterozygous mutant state, exacerbates the Abl mutant phenotypes (Henkemeyer et al., 1987). By characterizing such dosage-sensitive genes which can modify the Abl mutant phenotypes, a number of key proteins that function in the Abl pathway have since been identified. In addition to Disabled (a PTB domain–containing Abl substrate), mutations in Enabled (a proline-rich, tyrosine phosphorylated protein that binds Abl, Profilin, and actin), Dlar (a transmembrane tyrosine phosphatase), Trio (a Rac guanine nucleotide exchange factor), Chickadee (Profilin), Armadillo (-catenin), and Notch (a transmembrane receptor) have all been shown to modify the Abl mutant phenotypes (Lanier and Gertler, 2000). Further expanding the realm of Abl, Disabled and Enabled have themselves been implicated in important functions in flies and mammals, including the Reelin pathway, which is important for the normal layering of cortical neurons (Cooper and Howell, 1999). Many of the pathways these molecules function in are also implicated in Cdk5/p35 signaling, most notably in cadherin–catenin signaling and in cortical layering (see below). Thus, even without this new molecular connection between Abl and Cdk5, the two were already poised to converge. What is the purpose of Cables’ ability to bridge Cdk5 with Abl? All three proteins colocalize within cortical axons, particularly in their growth cones. One interesting possibility is that Cables and Abl both function as adaptor or scaffolding proteins to bind to Cdk5 and control its subcellular location in the neuron. Evidence for a noncatalytic adaptor-like function for Abl can be found in Drosophila, where it was demonstrated that the kinase activity of Abl was not needed to rescue the mutant phenotypes (Henkemeyer et al., 1990). Instead, it was found that two noncatalytic domains, one N terminal and the other C terminal, were absolutely essential for
Neuron 544
normal Abl function and that this function was somehow related to the ability of the Abl protein to properly localize within the axons of the embryonic CNS. Interestingly, it appears that in addition to the Abl SH3 domain, Cables also binds to two other regions of the Abl protein, one of which is the C-terminal domain and the other undefined (presumably the N terminus or the kinase domain). It may, therefore, be possible that the trimolecular interaction between Abl, Cables, and Cdk5 is required for the proper localization of the complex within the cell (namely the cytoskeleton), which could lead to the efficient phosphorylation of the Abl and Cdk5/p35 substrates that affect cytoskeletal dynamics. It will be interesting to determine whether Abl tyrosine kinase activity is essential for Cdk5/p35 function and what affect N-terminal and C-terminal domain mutations of Abl might have on Cdk5 localization. A search of the Drosophila genome indicates that a Cables-related sequence is indeed present (GenBank accession number AAF58349). This will permit genetic studies in the fly to investigate if wild-type Abl protein is important for Cables and Cdk5 localization and whether mutations in Cables or Cdk5 modify the Abl mutant phenotypes (and vice versa). The idea that Abl in mammals might have an essential function in Cdk5 signaling that is independent of its tyrosine kinase activity and more consistent with it acting as an adaptor is supported by mouse knockout studies that showed a truncated Abl protein that retains its kinase activity fails to function in the mouse, just like the Drosophila studies showed (Schwartzberg et al., 1991). However, it is important to point out that Abl can phosphorylate Cdk5 on tyrosine residue 15, and this increases the catalytic activity of the Cdk5 kinase domain. To further complicate the matter, Cdk5 is only active when bound to p35, and presumably the Abl/Cables/Cdk5 complex excludes p35 from binding. Thus, perhaps Abl and Cables are both adaptors that help colocalize Cdk5 to the proper subcellular compartment, and the Abl catalytic domain functions, in a regulated fashion, to activate Cdk5 serine/threonine kinase activity by phosphorylation of residue 15. But at this step, Cdk5 would then have to leave the Abl/Cables complex and bind p35 such that it can then phosphorylate its own target proteins, like Pak1, Tau, or -catenin. Unfortunately, the mechanisms that lead to the activation of the Abl tyrosine kinase domain in vivo are still somewhat of a mystery (Plattner et al., 1999). Beyond potentially elucidating Abl’s role as an adaptor protein, the new connection between Cables and Cdk5 reinforces the idea that Abl has important roles in regulating the cytoskeleton. Several lines of evidence implicate Abl as a key regulator of axonogenesis and cortical neuron migration. For example, Abl may lie downstream of a signaling cascade involving Reelin, very low density lipoprotein receptors, and murine Disabled (Scrambler), which have all been shown to be essential in the formation of the cortical neuron layers (Cooper and Howell, 1999). Quite interestingly, Cdk5 and p35 mutant mice also show cortical layering defects, suggesting that Abl, Cables, and Cdk5/p35 may all play a role in some aspect of Reelin signaling. To begin to uncover potential roles for Cables in cytoskeletal events, Zukerberg et al. (2000) established a neurite outgrowth assay using cultured cortical neurons. They found that
ectopic Cables expression inhibited neurite outgrowth, while ectopic Abl had the opposite effect and resulted in the elaboration of longer neurites. Although it is unclear exactly what these in vitro assays mean for the function of these molecules in vivo during brain development, they do provide a system to evaluate potentially important biological activities. For instance, it might be possible to elucidate the roles of Trio, Enabled, and Profilin with similar in vitro assays, particularly as they relate to Abl and Cables and ultimately in their potential regulation of actin and cytoskeletal dynamics at the growth cone. If we stop and try to connect the dots, it is clear that Abl and its partners have multiple roles in neuronal migration and axonal patterning, with a sum that points to Abl’s most physiologically relevant function being the regulation of cytoskeletal events. This new connection of Abl, Cables, and Cdk5 adds yet another potential mechanism by which diverse signals are integrated within the cell to control its architecture. The merging of Abl and Cdk5 is the latest in the ongoing search for an understanding of the signaling networks and protein– protein interactions that govern cytoskeletal dynamics. Clearly, this story is not over and much more research will be required to elucidate the biology of this intriguing protein in attempts to understand why all these “cables” lead to Abl. Chad A. Cowan and Mark Henkemeyer Center for Developmental Biology University of Texas Southwestern Medical Center Dallas, Texas 75235 Selected Reading Cooper, J.A., and Howell, B.W. (1999). Cell 97, 671–674. Henkemeyer, M.J., Gertler, F.B., Goodman, W., and Hoffmann, F.M. (1987). Cell 51, 821–828. Henkemeyer, M., West, S.R., Gertler, F.B., and Hoffmann, F.M. (1990). Cell 63, 949–960. Lanier, L.M., and Gertler, F.B. (2000). Curr. Opin. Neurobiol. 10, 80–87. Lee, M.-S., Kwon, Y.T., Li, M., Peng, J., Friedlander, R.M., and Tsai, L.-H. (2000). Nature 405, 360–364. Lew, J., Beaudette, K., Litwin, C.M., and Wang, J.H. (1992). J. Biol. Chem. 267, 13383–13390. Lew, J., Huang, Q.Q., Qi, Z., Winkfein, R.J., Aebersold, R., Hunt, T., and Wang, J.H. (1994). Nature 371, 423–426. Meyerson, M., Enders, G.H., Wu, C.L., Su, L.K., Gorka, C., Nelson, C., Harlow, E., and Tsai, L.-H. (1992). EMBO J. 11, 2909–2917. Plattner, R., Kadlec, L., DeMali, K.A., Kazlauskas, A., and Pendergast, A.M. (1999). Genes Dev. 13, 2400–2411. Schwartzberg, P.L., Stall, A.M., Hardin, J.D., Bowdish, K.S., Humaran, T., Boast, S., Harbison, M.L., Robertson, E.J., and Goff, S.P. (1991). Cell 65, 1165–1175. Tsai, L.-H., Delalle, I., Caviness, V.S., Chae, T., and Harlow, E. (1994). Nature 371, 419–423. Zukerberg, L.R., Patrick, G.N., Nikolic, M., Humbert, S., Wu, C.-L., Lanier, L.M., Gertler, F.B., Vidal, M., Van Etten, R.A., and Tsai, L.-H. (2000). Neuron 26, this issue, 633–646.