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Cat and mouse: viral cyclins and CDKs
276
News & Comment
TRENDS in Biochemical Sciences Vol.27 No.6 June 2002
Journal Club
Cat and mouse: viral cyclins and CDKs The progression of mammalian cells through the successive phases that lead to their final division into two daughter cells is a frequent event (in an adult human, millions of cells are manufactured each second); it must, therefore, be carefully regulated. The cyclic assembly of CDKs (cyclin-dependent kinases) with their cyclin partners regulates the cell-cycle by inducing appropriate downstream signals. Indeed, the binding of a cyclin molecule to its cognate CDK induces several conformational changes in the active site of the CDK, converting it to an active Ser/Thr kinase. In this paper, Schulze-Gahmen and Kim describe the crystal structure of an active complex between human CDK6 (a CDK that specifically regulates the G1 and G1/S mitotic transitions) and a cyclin from a herpes virus (Vcyclin) [1]. Although the overall structure of this complex is similar to that between CDK2 and cyclin A [2], some unique features emerge that suggest differences in the activation mechanism used by Vcyclin and its cellular counterparts. One peculiar feature of the CDK6–Vcyclin
complex stems from an N-terminal extension of Vcyclin. Although this region is mobile in the free form of Vcyclin, it becomes ordered upon interaction with CDK6, forming one β strand and two α helices. Moreover, this extension harbours several residues that wrap around the T-loop of CDK6 – the location of the Thr177CDK6 phosphorylation site (Thr160 in CDK2). As a consequence, the T-loop of CDK6 in the unphosphorylated CDK6–Vcyclin complex adopts a conformation that is similar to that of the phosphorylated CDK2 in complex with cyclin A. Given that the α helix PLSTIRE of CDK6 (corresponding to α helix PSTAIRE in CDK2) moves into the catalytic cleft, thereby bringing several catalytic residues into position, the active site of CDK6 appears fully open, an observation that is consistent with the high kinase activity of CDK6 both in its phosphorylated and unphosphorylated states. Finally, the interaction surface between Vcyclin and CDK6 is significantly larger than those seen for the cellular complexes. The authors suggest that the additional
contacts seen in the CDK6–Vcyclin complex make it resistant to cyclin-dependent inhibitory proteins. Infection by herpes viruses is characterized by the establishment of a latent infection, the expression of viral proteins being reduced to a few genes. Much effort is underway to elucidate the roles of the different proteins involved in the disruption of viral latency and in viral activation, which thereby lead to disease and cancer. This paper provides a beautiful example of the ‘cat-and-mouse game’ played by genes, viruses and cells. It will also stimulate further research aimed at manipulating signalling pathways using naturally occuring viral substitutes and analogues. 1 Schulze-Gahmen, U. and Kim, S-H. (2002) Structural basis for CDK6 activation by a virusencoded cyclin. Nat. Struct. Biol. 9, 177–181 2 Jeffrey, P.D. et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclinA–CDK2 complex. Nature 376, 313–320
Julien Lescar [email protected]
Caveolins in sickness and in health Caveola is certainly a magic word in today’s molecular cell biology, having the capacity to attract cell signalling, membrane traffic, and protein and lipid scientists simultaneously. ‘Lipid rafts’ and ‘detergent-resistant domains’ are often (and inaccurately) used as synonyms, but the term ‘caveola’ specifically defines the flask-shaped recesses that are found in the plasma membrane of some cells. Members of the caveolin protein family are typically associated with a subset of these membrane pits, which are, in addition, enriched in sphingolipids and cholesterol. Caveolins are small proteins with a remarkable capacity to bind cholesterol as well as a whole series of proteins involved in signal transduction pathways, such as trimeric G protein subunits, Src kinases and Raf. The paper by Carozzi et al. [1] deserves our attention because of its effort to relate the above data to certain forms of muscular dystrophy, namely Limb Girdle Muscular Dystrophy type 1C or hereditary rippling muscle disease. Caveolin-3 is the member of http://tibs.trends.com
the caveolin family that is found in the skeletal and cardiac muscle caveolae. To date, six caveolin-3 mutations (one microdeletion and five point mutations) have been found to be associated with the above pathologies. The authors have studied the capacity of some of these mutants to activate Raf in fibroblasts: they transfected BHK cells with activated H- or K-Ras (G12V), Raf and caveolin-3 mutant constructs, and immunoblotted for Ras and Raf-1. One of the point mutants, caveolin-3-C71W specifically inhibited signalling by activated H-Ras, but not by K-Ras. The same effect was seen in a different Ras assay system – neurite outgrowth in undifferentiated PC12 cells. Moreover, electron and confocal microscopy demonstrated that the C71W mutant colocalized with wild-type caveolin in BHK cells and differentiating muscle cells. Finally, increasing cholesterol levels in the BHK cells (by incubation with a cyclodextrin– cholesterol mixture) relieved the inhibition of H-Ras signalling caused by caveolin-3-C71W.
These data are interesting both from the point of view of caveolin–lipid interactions and from that of human pathology. Caveolins have been proposed to regulate the availability of cholesterol for the formation of raft domains, but the molecular details of the interaction of caveolins with cholesterol are still unknown. Expression of the mutant, but not of the wild-type, caveolin-3 caused a shift of the raft marker GFP-tH to denser membrane fractions, perhaps indicating a decreased cholesterol concentration in the raft domains. Carozzi et al. speculate that the C71W mutation increases the affinity of caveolin-3 for cholesterol, thus decreasing the availability of this lipid for inclusion in rafts. Studies with other C71 mutants of caveolin-3 show that it is the presence of tryptophan, rather than the loss of cysteine, that is important for the inhibitory effect of the mutant. Tryptophan has a high affinity for the membrane–water interface, and this might be related to the proposed increased affinity of the mutant for cholesterol.
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Biochemical Sciences Vol.27 No.6 June 2002
Journal Club
Cat and mouse: viral cyclins and CDKs The progression of mammalian cells through the successive phases that lead to their final division into two daughter cells is a frequent event (in an adult human, millions of cells are manufactured each second); it must, therefore, be carefully regulated. The cyclic assembly of CDKs (cyclin-dependent kinases) with their cyclin partners regulates the cell-cycle by inducing appropriate downstream signals. Indeed, the binding of a cyclin molecule to its cognate CDK induces several conformational changes in the active site of the CDK, converting it to an active Ser/Thr kinase. In this paper, Schulze-Gahmen and Kim describe the crystal structure of an active complex between human CDK6 (a CDK that specifically regulates the G1 and G1/S mitotic transitions) and a cyclin from a herpes virus (Vcyclin) [1]. Although the overall structure of this complex is similar to that between CDK2 and cyclin A [2], some unique features emerge that suggest differences in the activation mechanism used by Vcyclin and its cellular counterparts. One peculiar feature of the CDK6–Vcyclin
complex stems from an N-terminal extension of Vcyclin. Although this region is mobile in the free form of Vcyclin, it becomes ordered upon interaction with CDK6, forming one β strand and two α helices. Moreover, this extension harbours several residues that wrap around the T-loop of CDK6 – the location of the Thr177CDK6 phosphorylation site (Thr160 in CDK2). As a consequence, the T-loop of CDK6 in the unphosphorylated CDK6–Vcyclin complex adopts a conformation that is similar to that of the phosphorylated CDK2 in complex with cyclin A. Given that the α helix PLSTIRE of CDK6 (corresponding to α helix PSTAIRE in CDK2) moves into the catalytic cleft, thereby bringing several catalytic residues into position, the active site of CDK6 appears fully open, an observation that is consistent with the high kinase activity of CDK6 both in its phosphorylated and unphosphorylated states. Finally, the interaction surface between Vcyclin and CDK6 is significantly larger than those seen for the cellular complexes. The authors suggest that the additional
contacts seen in the CDK6–Vcyclin complex make it resistant to cyclin-dependent inhibitory proteins. Infection by herpes viruses is characterized by the establishment of a latent infection, the expression of viral proteins being reduced to a few genes. Much effort is underway to elucidate the roles of the different proteins involved in the disruption of viral latency and in viral activation, which thereby lead to disease and cancer. This paper provides a beautiful example of the ‘cat-and-mouse game’ played by genes, viruses and cells. It will also stimulate further research aimed at manipulating signalling pathways using naturally occuring viral substitutes and analogues. 1 Schulze-Gahmen, U. and Kim, S-H. (2002) Structural basis for CDK6 activation by a virusencoded cyclin. Nat. Struct. Biol. 9, 177–181 2 Jeffrey, P.D. et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclinA–CDK2 complex. Nature 376, 313–320
Julien Lescar [email protected]
Caveolins in sickness and in health Caveola is certainly a magic word in today’s molecular cell biology, having the capacity to attract cell signalling, membrane traffic, and protein and lipid scientists simultaneously. ‘Lipid rafts’ and ‘detergent-resistant domains’ are often (and inaccurately) used as synonyms, but the term ‘caveola’ specifically defines the flask-shaped recesses that are found in the plasma membrane of some cells. Members of the caveolin protein family are typically associated with a subset of these membrane pits, which are, in addition, enriched in sphingolipids and cholesterol. Caveolins are small proteins with a remarkable capacity to bind cholesterol as well as a whole series of proteins involved in signal transduction pathways, such as trimeric G protein subunits, Src kinases and Raf. The paper by Carozzi et al. [1] deserves our attention because of its effort to relate the above data to certain forms of muscular dystrophy, namely Limb Girdle Muscular Dystrophy type 1C or hereditary rippling muscle disease. Caveolin-3 is the member of http://tibs.trends.com
the caveolin family that is found in the skeletal and cardiac muscle caveolae. To date, six caveolin-3 mutations (one microdeletion and five point mutations) have been found to be associated with the above pathologies. The authors have studied the capacity of some of these mutants to activate Raf in fibroblasts: they transfected BHK cells with activated H- or K-Ras (G12V), Raf and caveolin-3 mutant constructs, and immunoblotted for Ras and Raf-1. One of the point mutants, caveolin-3-C71W specifically inhibited signalling by activated H-Ras, but not by K-Ras. The same effect was seen in a different Ras assay system – neurite outgrowth in undifferentiated PC12 cells. Moreover, electron and confocal microscopy demonstrated that the C71W mutant colocalized with wild-type caveolin in BHK cells and differentiating muscle cells. Finally, increasing cholesterol levels in the BHK cells (by incubation with a cyclodextrin– cholesterol mixture) relieved the inhibition of H-Ras signalling caused by caveolin-3-C71W.
These data are interesting both from the point of view of caveolin–lipid interactions and from that of human pathology. Caveolins have been proposed to regulate the availability of cholesterol for the formation of raft domains, but the molecular details of the interaction of caveolins with cholesterol are still unknown. Expression of the mutant, but not of the wild-type, caveolin-3 caused a shift of the raft marker GFP-tH to denser membrane fractions, perhaps indicating a decreased cholesterol concentration in the raft domains. Carozzi et al. speculate that the C71W mutation increases the affinity of caveolin-3 for cholesterol, thus decreasing the availability of this lipid for inclusion in rafts. Studies with other C71 mutants of caveolin-3 show that it is the presence of tryptophan, rather than the loss of cysteine, that is important for the inhibitory effect of the mutant. Tryptophan has a high affinity for the membrane–water interface, and this might be related to the proposed increased affinity of the mutant for cholesterol.
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.