Nervous System-Specific Proteins
24. 25. 26. 27.
C., Huang, K.-S., I’ratt, L)., Wachter, I,., Hession, C., Frey. A. Z. & Wallner. H. P. ( 1 988)J. Biol. Chem. 263, 10790- 108 1 1 Schlaepfer, L). L)., Jones, J. & Haigler, H. T. (1092) Biochemistry 3 1, 1886- 1891 Mochly-Kosen. I)., Khaner, H., Lopez, J. & Smith, H. I,. (1001) J. Hiol. Chem. 266, 14866 Morgan, A. & Hurgoyne, K. 1). (1992) Nature (London)355, X33-836 Kosenfeld. €3. G. C., Sanborn, H. & 1,oose-Mitchell, I).
(1991) Proceedings FASEH Conf. Atlanta, Georgia, Abstr. no. 2692, FASER J. p. A834 28. Nielsen, P. J. (1991) Hiochini. Hiophys. Acta 1088, 425-428 29. Swanson, K. L). & Ganguly, K. (1902) Gene 113, 183-190 30. van Heusden, G. P. H., Wenzel, T. J., Lagendijk,E. L., de Steensma, H. Y. & van den Berg, J. A. (1992) FEHS Lett. 302, 145-1 50 ~~
Received 16 April 1992
n-Chimaerin and neuronal signal transduction mechanisms Louis Lim Department of Neurochemistry, Institute of Neurology, I Wakefield Street, London WC I N I PJ, U.K. and Institute of Molecular and Cell Biology, National University of Singapore, Singapore 05 I I .
Introduction
has 48% identity to one half ( C l b ) of a duplicated sequence in the C1 regulatory region of PKC [4]. Binding of phorbol esters (DAG analogues) results in activation of PKC activity. The C-terminal domain has 42% identity over 171 amino acids to the C-terminal region of the product of the breakpoint cluster region gene (HCR) on chromosome 22, which is involved in the Philadelphia chromosome translocation associated with chronic myelogenous leukaemia [ 5 I . This reciprocal chromosomal (22:9) translocation generates a hybrid protein in which the Nterminal of HCR is fused to the kinase domain of abl with consequent activation of the kinase [6, 71.
In neurones, the binding by specific surface receptors of neurotransmitters or growth factors triggers off multi-component intracellular signalling pathways. For example, ligand-receptor binding leads to activation of membrane-bound phospholipase C which generates diacylglycerol (DAG). This second messenger in turn activates the key signal transduction enzyme protein kinase C (PKC) [ 11 whose phosphorylating activity towards various substrate proteins sets off a cascade of events. Other kinases, including receptor tyrosine kinases and also G-proteins, may be components of different signalling pathways, which can result in changes in gene transcription, cytoskeletal architecture and cellular metabolism [ 2 ]. The precise molecular interactions involved and the relationships between different pathways are the subject of much current research. It is well established that more genes are expressed in the brain than in any other tissues. Some of the genes uniquely expressed in the brain would be expected to subserve specialized function, particularly in neurones. Our research concerns the functional characterization of one such gene, encoding a protein we have termed n-chimaerin which may participate in novel signal transduction pathways involving DAG, whose only known receptor hitherto was PKC.
The bacterially expressed n-chimaerin (and the related C-terminal domain of HCR) acts as a GTPase activating protein (GAP) for p21rac [ l l ] . The The receptor activity lies within the cysteine-rich domain of the N-terminal which contains the concensus sequence €IX12CX2CX13CX2CX,HX2CX7C, also present in PKC. The binding affinity in vitro is similar for both, being in the nx,i range. Mutation of cysteine residues in either protein abolishes phorbol ester binding [9, 101. This binding activity is zincdependent [ l o ] .
n-Chimaerin contains two separate domains
n-Chimaerin functions as a GTPase activating protein
n-Chimaerin, the protein encoded by an mRNA expressed in neurones, consists of separate domains structurally related to two different protein families [ 31. The N-terminal cysteine-rich domain
The bacterially expressed n-chimaerin (and the related C-terminal domain of RCR) acts as a GTPase activating protein (GAPS) for p2lrac [ l l ] . The latter is a member of the ras superfamily of small GTP-binding proteins involved in various fundamental cellular processes including cell growth, differentiation and intracellular trafficking. The ras-like proteins have been implicated as central compo-
Abbreviations used: HCK, product of the breakpoint cluster region gene; DAG, diacylglycerol;GAP, GTPaseactivating protein; PKC, protein kinase C.
n-Chimaerin functions as a GTPase activating protein
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612
nents in cell signalling pathways, acting downstream of membrane receptors and functioning as molecular switches. All these p21 s possess intrinsic GTPase activity and cycle between GTP-bound (‘on’) and GDP-bound (‘off) states. The GTP-GDP cycle is regulated by several interacting proteins which include not only GAPs, but also exchange proteins and dissociation inhibitors [ 121. The GAP activity of n-chimaerin resides in its C-terminus. Two GAPs that down-regulate p21 ras, p 120 rasGAP [13] and NF1-GAP, the product of the gene involved in neurofibromatosis [ 14-16], have different affinities for p21 ras and are differentially regulated by lipids [ 17, 181. They may also have separate effector roles in addition to modulating intrinsic p2 1 YUS GTPase activity [ 121. For instance, p120 rusGAP has been shown to inhibit in vitro the opening of muscarinic receptor-coupled [K ] channels [lOl. The protein p21rac is a member of the rho subfamily thought to be involved in actin polymerization and cytoskeletal organization [20]. It is possible that n-chimaerin may have other cellular roles in adition to its racCAP activity, which results in down-regulation of p2 1 rac. +
Modulation of n-chimaerin GAP activity The presence in n-chimaerin of a phorbol esterbinding domain suggests that its p21rac GAP activity could be regulated in neurones by receptorstimulated generation of DAG. Using preparations of recombinant n-chimaerin, the $1 rac GAP activity has been found to be modulated by lipids known to act on PKC (S. Ahmed & J. Lee, unpublished work). Stimulation by phospholipids requires the presence of the N-terminal (putative regulatory) domain, and synergism between phospholipids and phorbol esters in stimulating GAP activity was observed.
Neuronal expression of n-chimaerin Polyclonal antibodies raised against TrpE-nchimaerin fusion proteins detect a 45 k n a protein specifically in brain and testis, with other minor components being present in brain (C. Monfries, unpublished work), a distribution consistent with expression of the mRNA 131. In the brain, nchimaerin mRNA is enriched in areas associated with learning and memory, especially in the hippocampus and cortex, with a specifically neuronal distribution demonstrated by in sztu hybridization (G. Michael, unpublished work). In the cerebellum the mRNA is restricted to Purkinje neurones. Interestingly, an avian n-chimaerin homologue with 96%
Volume 20
identity in amino acid sequence has been isolated, the mRNA for which is enriched in canary brain in areas associated with song control and neuronal plasticity [ 2 1 1.
Phenotypic effects of n-chimaerin expression The expression of stably transfected human nchimaerin cDNA (or the highly conserved rat cDNA) in neuronal cell lines resulted in phenotypic changes (R. Kozma & A. Hest, unpublished work). Neuroblastoma NIE-115 cells synthesized a 20 kI>a protein (p20”‘), detected by immunoblotting, in response to the transfected cIINAs. This is smaller than predicted from the full-length cDNAs, despite their giving rise to an appropriately sized mRNA in these cells. The p20”‘ contains the C-terminal (GAP) but not the N-terminal domain, as determined by immunological analysis with selective antibodies. Nevertheless, transiently transfected COS cells can express a 38 kI)a (full length) protein. It is not clear whether the truncated protein. which exhibits racGAP activity, found in neuroblastoma cells arises from proteolytic cleavage (as occurs with certain forms of I’KC) or alternative translation initiation (e.g. liver-enriched transcriptional activator protein; see [22]). The neuroblastoma cells expressing ~320‘’~ exhibit a different morphology and a reduced ability to differentiate in response to appropriate stimuli. Actin components of the cytoskeleton appear to be involved. These phenotypic changes in response to overexpressed n-chimaerin racGAP domain suggest that nchimaerin may function as a regulator of p21 rac activities associated with actin rearrangements and cytoskeletal organization [ 23 1.
n-Chimaerin is a member of a new GAP family n-Chimaerin, HCR and rhoGAI-’ can be considered as prototypes of a new GAP family [ 1 11. There are other proteins containing regions with sequence similarity to the GAP domain, including products of the yeast bud emergence genes HEM 2, HEM 3 [ 241 and 85 kDa proteins associated with phosphatidylinositol-3 kinase [25]; none of these have as yet been reported to have GAP activity, however. Our recent work has shown that the related C-terminal of REM 3 possesses rucGAI’ activity (C. Monfries & A. Bender, unpublished work). The sequence divergence of these proteins, including n-chimaerin and RCR, outside of the rucCAP domain, probably represents diverse regulatory domains that are responsive to different modulatory agents as well as
Nervous System-Specific Proteins
separate effector functions. HCR may have a multiplicity of roles, as it also contains various other activities, including that of a serine/threonine kinase 1261. IJsing an overlay assay to detect other members of this new GAP family, diverse GAPs with differing specificities towards the various members of the rho subfamily have been identified in a variety of tissues (E. Manser & T. Leung, unpublished work). Some of these new GAPs act on at least two members of the rho subfamily members, which include rho, rac and CDC42. This versatility niay be responsible for cross-talk between signalling pathways involving different GAPs effective towards the same p21 and/or between different p2 1s affected by the same GAP.
3.
4.
5. 6.
7. 8.
9.
Conclusion n-Chiniaerin is a specifically neuronal racGAP with a unique regulatory domain. The phorbol ester/ I>AC-binding domain may enable its GAP activity to be modulated in response to membrane-receptor-stimulated generation of DAG, in an analogous manner to PKC (previously the only known phorbol ester receptor). In neurones both n-chimaerin and PKC may be able to respond to the same stimulus either as components of the same signalling cascade or in divergent signalling pathways. In this context it is of interest that in brain areas involved in learning and memory processes (where n-chimaerin mRNA is highly expressed), PKC undergoes distributional changes after discrimination learning [27. 281. n-Chimaerin is clearly implicated in neuronal signal transduction with a possible involvement in p2 1-mediated neuronal cytoskeletal organization. Interestingly we have also found variants of n-chimaerin with alternative Nterminals (C. Hall & W.-C. Sin, unpublished work), which may subserve other specialized functions related to their binding to different cellular components. IIow these functions relate to the role of chimaerin in down-regulating the activities of the p21 rho subfamily as molecular switches remains to be determined.
10. 11.
12. 13.
14.
15.
16.
17.
18.
19.
20. The research on n-chimaerin summarized above represents published and unpublished work of laboratory colleagues in London and Singapore, whose invaluable contributions are gratefully acknowledged. I thank Alan Fiall for introducing us to the intricacies of the YUS superfamily and the Glaxo-Singapore Research Fund for its support. 1. Nishizuka. Y. (1988) Nature (London) 334, 661-665 2. Cantley, I,. C., Auger, K. K., Carpenter, C.,
21. 22. 23.
24. 25.
Duckworth, H., Graziani, A., Kapeller, K. & Soltoff, S. (199 1) Cell 64,28 1-302 Hall, C., Monfries, C., Smith, I’.? Lim, H. H., Kozma, K., Ahmed, S., Vanniasingham, V., Leung, T. & I i m , I,. (1990) J. Mol. Hiol. 211, 11-16 Parker, P. J., Coussens, I,., Totty, N., Khee, I,., Young, S., Chen, E., Stabel, S., Waterfield, M. I). & lillrich, A. (1986) Science 233, 853-859 Heisterkamp, N., Stam, K., Groffen, J., d e Klein, A. & Grosveld, G. (1985) Nature (London) 315,758-701 Konopka, J. H., Watanabe, S. M. & Witte, 0. N. (1984) Cell 37, 1035-1041 McWhiter, J. K. & Wang, W. Y. (1991) J. Mol. ljiol. 11, 1553-1565 Ahmed, S., Kozma, K., Monfries, C., Hall, C., Lim, H. H., Smith, 1’. & I h , I,. (1990) Riochem. J. 272, 767-773 Ono, Y., Fujii, T.. lgarashi, K., Kuno, T., Tanaka, C., Kikkawa, U. & Nishizuka, Y. (1989) I’roc. Natl. Acad. Sci. U.S.A. 86,4868-4871 Ahmed, S., Kozma, K., Lee, J., Monfries, C., Harden, N.& Lim, L. (1991) Hiochem. J. 280,233-241 Diekmann, D., Brill, S., Garrett, M. D., Totty, N., Hsuan, J.) Monfries, C., Hall, C., Lim, I,. & Hall, A. (1991) Nature (London) 351, 400-402 Hollag, G. & McCormick, F.(1991) Annu. Kev. Cell Hiol. 7.601-632 Vogel, U. S., Dixon, K. A. F., Schaber, M. 11.. Iliehl, K. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S. & Gibbs, J. (1988) Nature (London) 335, 90-93 Hallester, R., Marchuk, D.,Hoguski, M., Saulino, A., Letcher, R., Wigler, M. & Collins, F. (1988) Cell 53, 85 1-859 Martin, G. A,, Viskochil, L)., Hollag, G., McCabe, 1’. C., Crosier, W . J.? Haubruck, H., Conroy, I,., Clark, K., O’Connell, I’., Cawthorn, K. hl., Innis, M. A. & McCormick, F. (1 990) Cell 63,843-848 Xu, G., Lin, H., Tanaka, K., Llunn, D., Wood, W., Gesteland, K., White, K., Weiss, K. & Tamanoi, F. (1990) Cell 63, 835-841 Hollag, G. & McCormick, F. (199 1) Nature (London) 351,576-579 Golubic, M., Tanaka, K.. Llobrowolski, S., Wood, I)., Tsai, M. W., Marshall, M., Tamanoi, F. & Stacey, D. W . (1991) EMHO J. 10,2897-2903 Yatani, A,, Okabe, K., I’olakis, P., Halenbeck, K., McCormick, F. & Brown, A. M. (1990) Cell 61, 769-776 Hall, A. (1990) Science 249,635-640 George, J. M. & Clayton, 1). F. (1992) Molec. Hrain Kes. 12,323-329 Descombes, 1’. & Schibler, U. (1991) Cell 67, 569-579 Moll, J. Sansig, G., Fattore, E. & van der I’utten, 11. (1991) Oncogene 6, 863-86h Fry, M. J. (1992) Curr. Hiol. 2,78-80 Otsu, M., Hiles, I.. Gout, I., Fry, M. J., Ruiz-Larrea, F.) I’anayotou, G., Thompson, A., Ilhand, R., Ilsuan, J., Totty, N., Smith, A. I)., Morgan, S. J., Courtneidge,
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S.A,, Parker, 1’. J. & Waterfield, M. L). (1991) Cell 65, 9 1- 104
614
26. Maru. Y. & Witte. 0.N. (1991) Cell 67,455-468 27. Olds. J., Anderson, M. I,., McPhie, D. I,.. Staten. 1,. L). & Alkon, I). 1,. (1 989) Science 245,866-869
28. Olds, J. I,., Golski, I. S.. McPhie. L). I,., Olton, Ll., Mishkin, M. & Alkon, .)L I,. (1990) J. Neurosci. 10.
3707-3713 Received 16 April 1992
Intermediate filament protein expression in differentiating Schwann cells Peter J. Brophy,* Bernadette M. Kelly and C. Stewart Gillespie Department of Biological and Molecular Sciences, University of Stirling, Stirling, Scotland FK9 4LA, U.K.
Introduction In most eukaryotic cells the cytoskeleton consists of microtubules (diameter 25 nm), microfilaments (diameter 5-7 nm) and intermediate filaments (IFs; diameter 10 nm). IFs are morphologically similar in most eukaryotic cell types but they show a wide heterogeneity in their polypeptide subunits and these have been divided into six classes: types I and I1 (keratins in epithelial cells), type I11 [vimentin, desmin, glial fibrillary acidic protein (GFAP), peripherin], type IV (the neurofilaments NF-I,, NF-M and NF-H of apparent molecular masses 68, 150 and 200 kD, respectively, and a-internexin), type V (nuclear lamins) and type VI (nestin) [ 1,2]. Because IF proteins are often specific to particular cell types, they have been used as differentiation markers and as such are useful tools in the study of cell differentiation as well as in tumour identification. All IF proteins share common structural features: they are rod-shaped and have a large, highly conserved central a-helical core domain of approximately 3 10 residues which is flanked by two non-helical terminal domains of variable length and sequence at the N- and C-terminal regions. Despite extensive knowledge of their amino acid and cDNA sequences, the physiological function of most IF proteins remains elusive. Their linkage to both nucleus and plasma membrane [3] suggests a general role in the spatial organization of the cytoplasm; alternatively, IFs may be involved either in the transport of macromolecules between the nucleoplasmic and cytoplasmic compartments or in the transduction of information from the cell periphery to the nucleus [4]. IFs are commonly remodelled during cell maturation which suggests that the IF network has a role in differentiation. Neuroepithelial stem cells
Abbreviations used: FCS, fetal calf serum; GalC. galactocerebroside; GFAP, glial fibrillary acidic protein; IF, intermediate filament; NF, neurofilament. *To whom correspondence should be addressed.
Volume 20
initially coexpress nestin and vimentin, but nestin is subsequently down-regulated and the type IV IF protein a-internexin appears. Alpha-internexin is in turn replaced by the type IV proteins NF-I,, NF-M and NF-H that are characteristic of mature neurons [2]. The phenomenon of altered IF protein expression during terminal differentiation is also evident in glia. Mature oligodendrocytes lack IF proteins but their progenitors possess vimentin [ 51. Similarly, vimentin is the IF protein of immature Schwann cells although, unlike in oligodendrocytes, it is retained in differentiated cells and a second IF protein, GFAP, appears in Schwann cells at E18; upon terminal differentiation of myelin-forming Schwann cells, the expression of GFAP is suppressed whilst it is retained in non-myelin-forming Schwann cells
[61. Axonal contact stimulates Schwann cell proliferation and basement membrane formation and is also critical for myelin synthesis [ 7 ] .Some of the results of axonal contact such as Schwann cell proliferation and the cell-surface expression of the glycolipids galactocerebroside (GalC) and sulphatide (recognized by the 0 4 monoclonal antibody) can be mimicked in vitro by cyclic AMP and its analogues [8]. Recently, Morgan et al. [9] showed that the myelin-forming phenotype (up-regulated Po and down-regulated Ran-1, A5E3 and GFAP) could be induced in quiescent dissociated Schwann dells by raising intracellular cyclic AMP levels. In contrast, 0 4 expression was equally inducible in dividing and non-dividing cells. W e are interested in the role of the cytoskeleton in the differentiation of myelin-forming cells and in how the cytoskeleton regulates the extension of myelin processes during axonal ensheathment [ lo]. In this article we will describe a Schwann cell IF protein that is expressed at an early stage in the differentiation programme of myelinforming Schwann cells. This protein, which we initially called p145, colocalizes with vimentin and is inducible in culture by cyclic AMP analogues. Immunological analysis suggested that p 145 might
24. 25. 26. 27.
C., Huang, K.-S., I’ratt, L)., Wachter, I,., Hession, C., Frey. A. Z. & Wallner. H. P. ( 1 988)J. Biol. Chem. 263, 10790- 108 1 1 Schlaepfer, L). L)., Jones, J. & Haigler, H. T. (1092) Biochemistry 3 1, 1886- 1891 Mochly-Kosen. I)., Khaner, H., Lopez, J. & Smith, H. I,. (1001) J. Hiol. Chem. 266, 14866 Morgan, A. & Hurgoyne, K. 1). (1992) Nature (London)355, X33-836 Kosenfeld. €3. G. C., Sanborn, H. & 1,oose-Mitchell, I).
(1991) Proceedings FASEH Conf. Atlanta, Georgia, Abstr. no. 2692, FASER J. p. A834 28. Nielsen, P. J. (1991) Hiochini. Hiophys. Acta 1088, 425-428 29. Swanson, K. L). & Ganguly, K. (1902) Gene 113, 183-190 30. van Heusden, G. P. H., Wenzel, T. J., Lagendijk,E. L., de Steensma, H. Y. & van den Berg, J. A. (1992) FEHS Lett. 302, 145-1 50 ~~
Received 16 April 1992
n-Chimaerin and neuronal signal transduction mechanisms Louis Lim Department of Neurochemistry, Institute of Neurology, I Wakefield Street, London WC I N I PJ, U.K. and Institute of Molecular and Cell Biology, National University of Singapore, Singapore 05 I I .
Introduction
has 48% identity to one half ( C l b ) of a duplicated sequence in the C1 regulatory region of PKC [4]. Binding of phorbol esters (DAG analogues) results in activation of PKC activity. The C-terminal domain has 42% identity over 171 amino acids to the C-terminal region of the product of the breakpoint cluster region gene (HCR) on chromosome 22, which is involved in the Philadelphia chromosome translocation associated with chronic myelogenous leukaemia [ 5 I . This reciprocal chromosomal (22:9) translocation generates a hybrid protein in which the Nterminal of HCR is fused to the kinase domain of abl with consequent activation of the kinase [6, 71.
In neurones, the binding by specific surface receptors of neurotransmitters or growth factors triggers off multi-component intracellular signalling pathways. For example, ligand-receptor binding leads to activation of membrane-bound phospholipase C which generates diacylglycerol (DAG). This second messenger in turn activates the key signal transduction enzyme protein kinase C (PKC) [ 11 whose phosphorylating activity towards various substrate proteins sets off a cascade of events. Other kinases, including receptor tyrosine kinases and also G-proteins, may be components of different signalling pathways, which can result in changes in gene transcription, cytoskeletal architecture and cellular metabolism [ 2 ]. The precise molecular interactions involved and the relationships between different pathways are the subject of much current research. It is well established that more genes are expressed in the brain than in any other tissues. Some of the genes uniquely expressed in the brain would be expected to subserve specialized function, particularly in neurones. Our research concerns the functional characterization of one such gene, encoding a protein we have termed n-chimaerin which may participate in novel signal transduction pathways involving DAG, whose only known receptor hitherto was PKC.
The bacterially expressed n-chimaerin (and the related C-terminal domain of HCR) acts as a GTPase activating protein (GAP) for p21rac [ l l ] . The The receptor activity lies within the cysteine-rich domain of the N-terminal which contains the concensus sequence €IX12CX2CX13CX2CX,HX2CX7C, also present in PKC. The binding affinity in vitro is similar for both, being in the nx,i range. Mutation of cysteine residues in either protein abolishes phorbol ester binding [9, 101. This binding activity is zincdependent [ l o ] .
n-Chimaerin contains two separate domains
n-Chimaerin functions as a GTPase activating protein
n-Chimaerin, the protein encoded by an mRNA expressed in neurones, consists of separate domains structurally related to two different protein families [ 31. The N-terminal cysteine-rich domain
The bacterially expressed n-chimaerin (and the related C-terminal domain of RCR) acts as a GTPase activating protein (GAPS) for p2lrac [ l l ] . The latter is a member of the ras superfamily of small GTP-binding proteins involved in various fundamental cellular processes including cell growth, differentiation and intracellular trafficking. The ras-like proteins have been implicated as central compo-
Abbreviations used: HCK, product of the breakpoint cluster region gene; DAG, diacylglycerol;GAP, GTPaseactivating protein; PKC, protein kinase C.
n-Chimaerin functions as a GTPase activating protein
I992
61 I
Biochemical Society Transactions
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nents in cell signalling pathways, acting downstream of membrane receptors and functioning as molecular switches. All these p21 s possess intrinsic GTPase activity and cycle between GTP-bound (‘on’) and GDP-bound (‘off) states. The GTP-GDP cycle is regulated by several interacting proteins which include not only GAPs, but also exchange proteins and dissociation inhibitors [ 121. The GAP activity of n-chimaerin resides in its C-terminus. Two GAPs that down-regulate p21 ras, p 120 rasGAP [13] and NF1-GAP, the product of the gene involved in neurofibromatosis [ 14-16], have different affinities for p21 ras and are differentially regulated by lipids [ 17, 181. They may also have separate effector roles in addition to modulating intrinsic p2 1 YUS GTPase activity [ 121. For instance, p120 rusGAP has been shown to inhibit in vitro the opening of muscarinic receptor-coupled [K ] channels [lOl. The protein p21rac is a member of the rho subfamily thought to be involved in actin polymerization and cytoskeletal organization [20]. It is possible that n-chimaerin may have other cellular roles in adition to its racCAP activity, which results in down-regulation of p2 1 rac. +
Modulation of n-chimaerin GAP activity The presence in n-chimaerin of a phorbol esterbinding domain suggests that its p21rac GAP activity could be regulated in neurones by receptorstimulated generation of DAG. Using preparations of recombinant n-chimaerin, the $1 rac GAP activity has been found to be modulated by lipids known to act on PKC (S. Ahmed & J. Lee, unpublished work). Stimulation by phospholipids requires the presence of the N-terminal (putative regulatory) domain, and synergism between phospholipids and phorbol esters in stimulating GAP activity was observed.
Neuronal expression of n-chimaerin Polyclonal antibodies raised against TrpE-nchimaerin fusion proteins detect a 45 k n a protein specifically in brain and testis, with other minor components being present in brain (C. Monfries, unpublished work), a distribution consistent with expression of the mRNA 131. In the brain, nchimaerin mRNA is enriched in areas associated with learning and memory, especially in the hippocampus and cortex, with a specifically neuronal distribution demonstrated by in sztu hybridization (G. Michael, unpublished work). In the cerebellum the mRNA is restricted to Purkinje neurones. Interestingly, an avian n-chimaerin homologue with 96%
Volume 20
identity in amino acid sequence has been isolated, the mRNA for which is enriched in canary brain in areas associated with song control and neuronal plasticity [ 2 1 1.
Phenotypic effects of n-chimaerin expression The expression of stably transfected human nchimaerin cDNA (or the highly conserved rat cDNA) in neuronal cell lines resulted in phenotypic changes (R. Kozma & A. Hest, unpublished work). Neuroblastoma NIE-115 cells synthesized a 20 kI>a protein (p20”‘), detected by immunoblotting, in response to the transfected cIINAs. This is smaller than predicted from the full-length cDNAs, despite their giving rise to an appropriately sized mRNA in these cells. The p20”‘ contains the C-terminal (GAP) but not the N-terminal domain, as determined by immunological analysis with selective antibodies. Nevertheless, transiently transfected COS cells can express a 38 kI)a (full length) protein. It is not clear whether the truncated protein. which exhibits racGAP activity, found in neuroblastoma cells arises from proteolytic cleavage (as occurs with certain forms of I’KC) or alternative translation initiation (e.g. liver-enriched transcriptional activator protein; see [22]). The neuroblastoma cells expressing ~320‘’~ exhibit a different morphology and a reduced ability to differentiate in response to appropriate stimuli. Actin components of the cytoskeleton appear to be involved. These phenotypic changes in response to overexpressed n-chimaerin racGAP domain suggest that nchimaerin may function as a regulator of p21 rac activities associated with actin rearrangements and cytoskeletal organization [ 23 1.
n-Chimaerin is a member of a new GAP family n-Chimaerin, HCR and rhoGAI-’ can be considered as prototypes of a new GAP family [ 1 11. There are other proteins containing regions with sequence similarity to the GAP domain, including products of the yeast bud emergence genes HEM 2, HEM 3 [ 241 and 85 kDa proteins associated with phosphatidylinositol-3 kinase [25]; none of these have as yet been reported to have GAP activity, however. Our recent work has shown that the related C-terminal of REM 3 possesses rucGAI’ activity (C. Monfries & A. Bender, unpublished work). The sequence divergence of these proteins, including n-chimaerin and RCR, outside of the rucCAP domain, probably represents diverse regulatory domains that are responsive to different modulatory agents as well as
Nervous System-Specific Proteins
separate effector functions. HCR may have a multiplicity of roles, as it also contains various other activities, including that of a serine/threonine kinase 1261. IJsing an overlay assay to detect other members of this new GAP family, diverse GAPs with differing specificities towards the various members of the rho subfamily have been identified in a variety of tissues (E. Manser & T. Leung, unpublished work). Some of these new GAPs act on at least two members of the rho subfamily members, which include rho, rac and CDC42. This versatility niay be responsible for cross-talk between signalling pathways involving different GAPs effective towards the same p21 and/or between different p2 1s affected by the same GAP.
3.
4.
5. 6.
7. 8.
9.
Conclusion n-Chiniaerin is a specifically neuronal racGAP with a unique regulatory domain. The phorbol ester/ I>AC-binding domain may enable its GAP activity to be modulated in response to membrane-receptor-stimulated generation of DAG, in an analogous manner to PKC (previously the only known phorbol ester receptor). In neurones both n-chimaerin and PKC may be able to respond to the same stimulus either as components of the same signalling cascade or in divergent signalling pathways. In this context it is of interest that in brain areas involved in learning and memory processes (where n-chimaerin mRNA is highly expressed), PKC undergoes distributional changes after discrimination learning [27. 281. n-Chimaerin is clearly implicated in neuronal signal transduction with a possible involvement in p2 1-mediated neuronal cytoskeletal organization. Interestingly we have also found variants of n-chimaerin with alternative Nterminals (C. Hall & W.-C. Sin, unpublished work), which may subserve other specialized functions related to their binding to different cellular components. IIow these functions relate to the role of chimaerin in down-regulating the activities of the p21 rho subfamily as molecular switches remains to be determined.
10. 11.
12. 13.
14.
15.
16.
17.
18.
19.
20. The research on n-chimaerin summarized above represents published and unpublished work of laboratory colleagues in London and Singapore, whose invaluable contributions are gratefully acknowledged. I thank Alan Fiall for introducing us to the intricacies of the YUS superfamily and the Glaxo-Singapore Research Fund for its support. 1. Nishizuka. Y. (1988) Nature (London) 334, 661-665 2. Cantley, I,. C., Auger, K. K., Carpenter, C.,
21. 22. 23.
24. 25.
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3707-3713 Received 16 April 1992
Intermediate filament protein expression in differentiating Schwann cells Peter J. Brophy,* Bernadette M. Kelly and C. Stewart Gillespie Department of Biological and Molecular Sciences, University of Stirling, Stirling, Scotland FK9 4LA, U.K.
Introduction In most eukaryotic cells the cytoskeleton consists of microtubules (diameter 25 nm), microfilaments (diameter 5-7 nm) and intermediate filaments (IFs; diameter 10 nm). IFs are morphologically similar in most eukaryotic cell types but they show a wide heterogeneity in their polypeptide subunits and these have been divided into six classes: types I and I1 (keratins in epithelial cells), type I11 [vimentin, desmin, glial fibrillary acidic protein (GFAP), peripherin], type IV (the neurofilaments NF-I,, NF-M and NF-H of apparent molecular masses 68, 150 and 200 kD, respectively, and a-internexin), type V (nuclear lamins) and type VI (nestin) [ 1,2]. Because IF proteins are often specific to particular cell types, they have been used as differentiation markers and as such are useful tools in the study of cell differentiation as well as in tumour identification. All IF proteins share common structural features: they are rod-shaped and have a large, highly conserved central a-helical core domain of approximately 3 10 residues which is flanked by two non-helical terminal domains of variable length and sequence at the N- and C-terminal regions. Despite extensive knowledge of their amino acid and cDNA sequences, the physiological function of most IF proteins remains elusive. Their linkage to both nucleus and plasma membrane [3] suggests a general role in the spatial organization of the cytoplasm; alternatively, IFs may be involved either in the transport of macromolecules between the nucleoplasmic and cytoplasmic compartments or in the transduction of information from the cell periphery to the nucleus [4]. IFs are commonly remodelled during cell maturation which suggests that the IF network has a role in differentiation. Neuroepithelial stem cells
Abbreviations used: FCS, fetal calf serum; GalC. galactocerebroside; GFAP, glial fibrillary acidic protein; IF, intermediate filament; NF, neurofilament. *To whom correspondence should be addressed.
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initially coexpress nestin and vimentin, but nestin is subsequently down-regulated and the type IV IF protein a-internexin appears. Alpha-internexin is in turn replaced by the type IV proteins NF-I,, NF-M and NF-H that are characteristic of mature neurons [2]. The phenomenon of altered IF protein expression during terminal differentiation is also evident in glia. Mature oligodendrocytes lack IF proteins but their progenitors possess vimentin [ 51. Similarly, vimentin is the IF protein of immature Schwann cells although, unlike in oligodendrocytes, it is retained in differentiated cells and a second IF protein, GFAP, appears in Schwann cells at E18; upon terminal differentiation of myelin-forming Schwann cells, the expression of GFAP is suppressed whilst it is retained in non-myelin-forming Schwann cells
[61. Axonal contact stimulates Schwann cell proliferation and basement membrane formation and is also critical for myelin synthesis [ 7 ] .Some of the results of axonal contact such as Schwann cell proliferation and the cell-surface expression of the glycolipids galactocerebroside (GalC) and sulphatide (recognized by the 0 4 monoclonal antibody) can be mimicked in vitro by cyclic AMP and its analogues [8]. Recently, Morgan et al. [9] showed that the myelin-forming phenotype (up-regulated Po and down-regulated Ran-1, A5E3 and GFAP) could be induced in quiescent dissociated Schwann dells by raising intracellular cyclic AMP levels. In contrast, 0 4 expression was equally inducible in dividing and non-dividing cells. W e are interested in the role of the cytoskeleton in the differentiation of myelin-forming cells and in how the cytoskeleton regulates the extension of myelin processes during axonal ensheathment [ lo]. In this article we will describe a Schwann cell IF protein that is expressed at an early stage in the differentiation programme of myelinforming Schwann cells. This protein, which we initially called p145, colocalizes with vimentin and is inducible in culture by cyclic AMP analogues. Immunological analysis suggested that p 145 might
