Guillermo de Cárcer | Spanish National Cancer Research Center (original) (raw)

Papers by Guillermo de Cárcer

Blood, Jan 16, 2015

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which ... more Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the Spindle Assembly Checkpoint (SAC), and can be rescued in the presence of SAC i...

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles du... more Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non-mitotic arrest in early embryos, we show here that the bi-allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi-and mono-polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid-gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1-heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small-molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.

Journal of Structural Biology, 1999

Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation,... more Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation, under the conventional fluorescence microscope, the confocal microscope, and the transmission electron microscope. The confocal microscopy study of transcription was combined with immunodetection of fibrillarin, a component of the RNP complex involved in the early processing of pre-rRNA. Superposition of transcription and fibrillarin images from the same optical section showed some small ''black holes'' in the nucleolus, around which a lateral and radial differentiation of labeling was observed: laterally, zones corresponding to transcription labeling alternated with zones of fibrillarin labeling; radially, areas of transcription gradually became areas of colocalization of transcription and fibrillarin, and, further outward, of fibrillarin alone, which occupied the major part of the labeled nucleolar area. Three-dimensional reconstruction of the nucleolar transcription labeling, from confocal optical sections, showed clusters of foci arranged around an area of low or no labeling. Thin labeled extensions, connecting single foci, were observed. Visualization of transcription at the ultrastructural level identified the black holes as fibrillar centers, in view of their size and the absence of labeling in them. In fact, most of the labeling was observed in discrete areas of the dense fibrillar component, near fibrillar centers, including the transition area between these two components. This observation was supported by a quantitative study. Otherwise, the outline of fibrillar centers did not appear entirely surrounded by particles, and a minor proportion of particles was detected dispersed throughout the dense fibrillar component. As a complementary study, the transcription factor upstream binding factor (UBF) and the protein NopA64, a plant nucleolin homologue, were immunolocalized. Small foci of UBF localization alone and other foci in which the two protein markers overlapped were observed. The outer areas of the nucleolus showed the exclusive presence of NopA64. Under the electron microscope, UBF labeling, quantitatively assessed, appeared as clusters of particles, most of them surrounding fibrillar centers. A graphic model is presented to give a molecular interpretation of these data. 1999 Academic Press

Current Medicinal Chemistry, 2007

Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in ... more Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in the progression throughout the cell division cycle. The cyclin-dependent kinase (CDK) family has received special attention due to their function as sensors of the mitogenic signals and their central role in cell proliferation. These kinases are frequently upregulated in human cancer most frequently due to overexpression of their cyclin partners or inactivation of the CDK inhibitors. A plethora of small-molecule CDK inhibitors have been characterized in the last years and some of them are currently under clinical development. Other serine-threonine protein kinases such as the Aurora proteins (mostly Aurora A and B) or Polo-like kinases (PLK1) are receiving increased attention as putative cancer targets. Other less studied mitotic kinases such TTK (MPS1), BUB and NEK proteins might also be relevant candidates as new targets of interest in cancer therapy since they play relevant roles on mitotic progression and the spindle checkpoint. Although targeting cell cycle kinases is an efficient procedure to arrest cell proliferation, the best strategy to potently and specifically inhibit tumor cell proliferation is not obvious yet. Thus, some cell cycle kinases may be of interest as targets to abrogate checkpoints and favor apoptotic cell death in tumor cells. New biochemical and genetic studies are required to clarify the use of these kinases as targets in new opportunities to improve cancer therapy.

Carcinogenesis, 2006

Abbreviations: APC/C, anaphase-promoting complex/cyclosome; CDK, cyclin-dependent kinase; FZR1, f... more Abbreviations: APC/C, anaphase-promoting complex/cyclosome; CDK, cyclin-dependent kinase; FZR1, fizzy-related 1; PLK1, polo-like kinase 1; SAC, spindle assembly checkpoint.

Cancer Research, 2004

We have shown previously that the molecular chaperone heat shock protein 90 (Hsp90) is required f... more We have shown previously that the molecular chaperone heat shock protein 90 (Hsp90) is required for a proper centrosome function. Indeed, this Hsp90 function seems to be reflected in Polo-like kinase stability. Inhibition of Hsp90 in HeLa cells results in cell cycle arrest either in G2 stage or at the metaphase-anaphase transition. Here, we show that this inhibition leads to inactivation of the anaphase-promoting complex or cyclosome by both dephosphorylation and induction of the spindle assembly checkpoint. Hsp90 inhibition compromises two of the main mitotic kinases, Polo-like kinase 1 (Plk1) and cdc2. Interestingly, this mitotic arrest does not occur in certain tumor cell lines where Hsp90 and Plk1 are not associated. Those cells are able to process mitosis successfully and have an active Plk1 despite Hsp90 inactivation. Therefore, it seems that Hsp90 regulates completion of mitosis depending on its association with Plk1.

Blood, Jan 16, 2015

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which ... more Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the Spindle Assembly Checkpoint (SAC), and can be rescued in the presence of SAC i...

The EMBO Journal, 2001

We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required t... more We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant Polo. We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.

Proceedings of the National Academy of Sciences, 1999

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mR... more An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase-PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of Ϸ145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

Proceedings of the National Academy of Sciences, 2007

Molecular and Cellular Biology, 2009

TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in ... more TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in telomere biology in the context of the organism is unclear. In this study, we generated mice with transgenic TRF1 expression targeted to epithelial tissues (K5TRF1 mice). K5TRF1 mice have shorter telomeres in the epidermis than wild-type controls do, and these are rescued in the absence of the XPF nuclease, indicating that TRF1 acts as a negative regulator of telomere length by controlling XPF activity at telomeres, similar to what was previously described for TRF2-overexpressing mice (K5TRF2 mice). K5TRF1 cells also show increased end-toend chromosomal fusions, multitelomeric signals, and increased telomere recombination, indicating an impact of TRF1 on telomere integrity, again similar to the case in K5TRF2 cells. Intriguingly, K5TRF1 cells, but not K5TRF2 cells, show increased mitotic spindle aberrations. TRF1 colocalizes with the spindle assembly checkpoint proteins BubR1 and Mad2 at mouse telomeres, indicating a link between telomeres and the mitotic spindle. Together, these results demonstrate that TRF1, like TRF2, negatively regulates telomere length in vivo by controlling the action of the XPF nuclease at telomeres; in addition, TRF1 has a unique role in the mitotic spindle checkpoint.

Molecular and Cellular Biology, 2011

Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the pol... more Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the polo box (PBD), involved in protein-protein interactions. Plk1 to Plk4 are involved in centrosome biology as well as the regulation of mitosis, cytokinesis, and cell cycle checkpoints in response to genotoxic stress. We have analyzed here the new member of the vertebrate family, Plk5, a protein that lacks the kinase domain in humans. Plk5 does not seem to have a role in cell cycle progression; in fact, it is downregulated in proliferating cells and accumulates in quiescent cells. This protein is mostly expressed in the brain of both mice and humans, and it modulates the formation of neuritic processes upon stimulation of the brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF)-Ras pathway in neurons. The human PLK5 gene is significantly silenced in astrocytoma and glioblastoma multiforme by promoter hypermethylation, suggesting a tumor suppressor function for this gene. Indeed, overexpression of Plk5 has potent apoptotic effects in these tumor cells. Thus, Plk5 seems to have evolved as a kinase-deficient PBD-containing protein with nervous system-specific functions and tumor suppressor activity in brain cancer.

Journal of Cell Science, 2010

Cancer Cell, 2010

Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against canc... more Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55a or B55d regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.

Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nuc... more Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nucleation of actin fibers. The clinical relevance of Brk1 is emphasized by correlative data showing that Von Hippel-Lindau (VHL) patients that also lose the BRK1 gene are protected against the development of tumors. This contrasts with recent evidence suggesting that the Wave complex may function as an invasion suppressor in epithelial cancers. Here, we show that the downregulation of Brk1 results in abnormal actin stress fiber formation and vinculin distribution and loss of Arp2/3 and Wave proteins at the cellular protrusions. Brk1 is required for cell proliferation and cell transformation by oncogenes. In addition, Brk1 downregulation results in defective directional migration and invasive growth in renal cell carcinoma cells as well as in other tumor cell types. Finally, genetic ablation of Brk1 results in dramatic defects in embryo compaction and development, suggesting an essential role for this protein in actin dynamics. Thus, genetic loss or inhibition of BRK1 is likely to be protective against tumor development due to proliferation and motility defects in affected cells. Cancer Res; 70(22); OF1-11.

Molecular and cellular biology, 1998

An increasing number of independent studies indicate that the atypical protein kinase C (PKC) iso... more An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of lambda/iotaPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both lambda/iotaPKC and zetaPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56(lck) SH2-interacting protein, as a molecule that interacts potently with the V1 domain of lambda/iotaPKC and, albeit with lower affinity, with zetaPKC. We also show in this study that ectopically expressed p62 col...

Blood, Jan 16, 2015

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which ... more Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the Spindle Assembly Checkpoint (SAC), and can be rescued in the presence of SAC i...

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles du... more Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non-mitotic arrest in early embryos, we show here that the bi-allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi-and mono-polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid-gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1-heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small-molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.

Journal of Structural Biology, 1999

Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation,... more Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation, under the conventional fluorescence microscope, the confocal microscope, and the transmission electron microscope. The confocal microscopy study of transcription was combined with immunodetection of fibrillarin, a component of the RNP complex involved in the early processing of pre-rRNA. Superposition of transcription and fibrillarin images from the same optical section showed some small ''black holes'' in the nucleolus, around which a lateral and radial differentiation of labeling was observed: laterally, zones corresponding to transcription labeling alternated with zones of fibrillarin labeling; radially, areas of transcription gradually became areas of colocalization of transcription and fibrillarin, and, further outward, of fibrillarin alone, which occupied the major part of the labeled nucleolar area. Three-dimensional reconstruction of the nucleolar transcription labeling, from confocal optical sections, showed clusters of foci arranged around an area of low or no labeling. Thin labeled extensions, connecting single foci, were observed. Visualization of transcription at the ultrastructural level identified the black holes as fibrillar centers, in view of their size and the absence of labeling in them. In fact, most of the labeling was observed in discrete areas of the dense fibrillar component, near fibrillar centers, including the transition area between these two components. This observation was supported by a quantitative study. Otherwise, the outline of fibrillar centers did not appear entirely surrounded by particles, and a minor proportion of particles was detected dispersed throughout the dense fibrillar component. As a complementary study, the transcription factor upstream binding factor (UBF) and the protein NopA64, a plant nucleolin homologue, were immunolocalized. Small foci of UBF localization alone and other foci in which the two protein markers overlapped were observed. The outer areas of the nucleolus showed the exclusive presence of NopA64. Under the electron microscope, UBF labeling, quantitatively assessed, appeared as clusters of particles, most of them surrounding fibrillar centers. A graphic model is presented to give a molecular interpretation of these data. 1999 Academic Press

Current Medicinal Chemistry, 2007

Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in ... more Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in the progression throughout the cell division cycle. The cyclin-dependent kinase (CDK) family has received special attention due to their function as sensors of the mitogenic signals and their central role in cell proliferation. These kinases are frequently upregulated in human cancer most frequently due to overexpression of their cyclin partners or inactivation of the CDK inhibitors. A plethora of small-molecule CDK inhibitors have been characterized in the last years and some of them are currently under clinical development. Other serine-threonine protein kinases such as the Aurora proteins (mostly Aurora A and B) or Polo-like kinases (PLK1) are receiving increased attention as putative cancer targets. Other less studied mitotic kinases such TTK (MPS1), BUB and NEK proteins might also be relevant candidates as new targets of interest in cancer therapy since they play relevant roles on mitotic progression and the spindle checkpoint. Although targeting cell cycle kinases is an efficient procedure to arrest cell proliferation, the best strategy to potently and specifically inhibit tumor cell proliferation is not obvious yet. Thus, some cell cycle kinases may be of interest as targets to abrogate checkpoints and favor apoptotic cell death in tumor cells. New biochemical and genetic studies are required to clarify the use of these kinases as targets in new opportunities to improve cancer therapy.

Carcinogenesis, 2006

Abbreviations: APC/C, anaphase-promoting complex/cyclosome; CDK, cyclin-dependent kinase; FZR1, f... more Abbreviations: APC/C, anaphase-promoting complex/cyclosome; CDK, cyclin-dependent kinase; FZR1, fizzy-related 1; PLK1, polo-like kinase 1; SAC, spindle assembly checkpoint.

Cancer Research, 2004

We have shown previously that the molecular chaperone heat shock protein 90 (Hsp90) is required f... more We have shown previously that the molecular chaperone heat shock protein 90 (Hsp90) is required for a proper centrosome function. Indeed, this Hsp90 function seems to be reflected in Polo-like kinase stability. Inhibition of Hsp90 in HeLa cells results in cell cycle arrest either in G2 stage or at the metaphase-anaphase transition. Here, we show that this inhibition leads to inactivation of the anaphase-promoting complex or cyclosome by both dephosphorylation and induction of the spindle assembly checkpoint. Hsp90 inhibition compromises two of the main mitotic kinases, Polo-like kinase 1 (Plk1) and cdc2. Interestingly, this mitotic arrest does not occur in certain tumor cell lines where Hsp90 and Plk1 are not associated. Those cells are able to process mitosis successfully and have an active Plk1 despite Hsp90 inactivation. Therefore, it seems that Hsp90 regulates completion of mitosis depending on its association with Plk1.

Blood, Jan 16, 2015

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which ... more Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the Spindle Assembly Checkpoint (SAC), and can be rescued in the presence of SAC i...

The EMBO Journal, 2001

We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required t... more We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant Polo. We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.

Proceedings of the National Academy of Sciences, 1999

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mR... more An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase-PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of Ϸ145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

Proceedings of the National Academy of Sciences, 2007

Molecular and Cellular Biology, 2009

TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in ... more TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in telomere biology in the context of the organism is unclear. In this study, we generated mice with transgenic TRF1 expression targeted to epithelial tissues (K5TRF1 mice). K5TRF1 mice have shorter telomeres in the epidermis than wild-type controls do, and these are rescued in the absence of the XPF nuclease, indicating that TRF1 acts as a negative regulator of telomere length by controlling XPF activity at telomeres, similar to what was previously described for TRF2-overexpressing mice (K5TRF2 mice). K5TRF1 cells also show increased end-toend chromosomal fusions, multitelomeric signals, and increased telomere recombination, indicating an impact of TRF1 on telomere integrity, again similar to the case in K5TRF2 cells. Intriguingly, K5TRF1 cells, but not K5TRF2 cells, show increased mitotic spindle aberrations. TRF1 colocalizes with the spindle assembly checkpoint proteins BubR1 and Mad2 at mouse telomeres, indicating a link between telomeres and the mitotic spindle. Together, these results demonstrate that TRF1, like TRF2, negatively regulates telomere length in vivo by controlling the action of the XPF nuclease at telomeres; in addition, TRF1 has a unique role in the mitotic spindle checkpoint.

Molecular and Cellular Biology, 2011

Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the pol... more Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the polo box (PBD), involved in protein-protein interactions. Plk1 to Plk4 are involved in centrosome biology as well as the regulation of mitosis, cytokinesis, and cell cycle checkpoints in response to genotoxic stress. We have analyzed here the new member of the vertebrate family, Plk5, a protein that lacks the kinase domain in humans. Plk5 does not seem to have a role in cell cycle progression; in fact, it is downregulated in proliferating cells and accumulates in quiescent cells. This protein is mostly expressed in the brain of both mice and humans, and it modulates the formation of neuritic processes upon stimulation of the brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF)-Ras pathway in neurons. The human PLK5 gene is significantly silenced in astrocytoma and glioblastoma multiforme by promoter hypermethylation, suggesting a tumor suppressor function for this gene. Indeed, overexpression of Plk5 has potent apoptotic effects in these tumor cells. Thus, Plk5 seems to have evolved as a kinase-deficient PBD-containing protein with nervous system-specific functions and tumor suppressor activity in brain cancer.

Journal of Cell Science, 2010

Cancer Cell, 2010

Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against canc... more Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55a or B55d regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.

Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nuc... more Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nucleation of actin fibers. The clinical relevance of Brk1 is emphasized by correlative data showing that Von Hippel-Lindau (VHL) patients that also lose the BRK1 gene are protected against the development of tumors. This contrasts with recent evidence suggesting that the Wave complex may function as an invasion suppressor in epithelial cancers. Here, we show that the downregulation of Brk1 results in abnormal actin stress fiber formation and vinculin distribution and loss of Arp2/3 and Wave proteins at the cellular protrusions. Brk1 is required for cell proliferation and cell transformation by oncogenes. In addition, Brk1 downregulation results in defective directional migration and invasive growth in renal cell carcinoma cells as well as in other tumor cell types. Finally, genetic ablation of Brk1 results in dramatic defects in embryo compaction and development, suggesting an essential role for this protein in actin dynamics. Thus, genetic loss or inhibition of BRK1 is likely to be protective against tumor development due to proliferation and motility defects in affected cells. Cancer Res; 70(22); OF1-11.

Molecular and cellular biology, 1998

An increasing number of independent studies indicate that the atypical protein kinase C (PKC) iso... more An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of lambda/iotaPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both lambda/iotaPKC and zetaPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56(lck) SH2-interacting protein, as a molecule that interacts potently with the V1 domain of lambda/iotaPKC and, albeit with lower affinity, with zetaPKC. We also show in this study that ectopically expressed p62 col...