Regulation of GRK2 by Mdm2 and the APC/C complex: a way to fine-tune dynamics and faithful
progression of the cell cycle
Clara Reglero Gómez
Madrid, 2017
Facultad de Ciencias
Departamento de Biología Molecular
CLARA REGLERO GÓMEZ
para optar al título de
DOCTORA POR LA UNIVERSIDAD AUTÓNOMA DE MADRID EN BIOQUÍMICA, BIOLOGÍA MOLECULAR, BIOMEDICINA Y
BIOTECNOLOGÍA
Directores de la tesis:
DRA. PETRONILA PENELA MÁRQUEZ DR. FEDERICO MAYOR MENÉNDEZ
Este trabajo ha sido realizado en el Departamento de Biología Mo- lecular de la Facultad de Ciencias de la Universidad Autónoma de
Madrid. Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM).
Este trabajo se ha llevado a cabo en el Centro de Biología Molecular Severo Ochoa (CSIC-UAM) bajo la dirección de Petronila Penela Márquez, Profesora Titular del Departamento de Biología Molecular de la Universidad Autónoma de Madrid, y de Federico Mayor Menéndez, Catedrático del Departamento de Biología Molecular de la Universidad Autónoma de Madrid.
La realización de esta Tesis ha sido posible gracias a fon- dos de Proyectos I+D+i correspondientes al Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Re- tos de la Sociedad del Ministerio de Economía y Competitivi- dad, y de la Fundación Severo Ochoa. La autora también ha recibido apoyo a través de becas de EMBO, IUBMB, FEBS y SEBBM para asistencia a congresos.
ABSTRACT...13
RESUMEN...17
INTRODUCTION...21
1. GPCRs and GRKs in cellular growth...23
2. GRK2 as a key node in signal transduction pathways related to the hallmarks of cancer....26
2.1. The complex GRK2 interactome: implications in cell metabolism, motility, proliferation and survival...27
2.2. Regulation of the activity and protein turnover of GRK2...30
2.3. GRK2 in cancer progression...33
3. The cell cycle progression...36
3.1. G1 phase...37
3.2. S phase...37
3.3. G2 phase...37
3.4. Mitosis...38
a) Mitotic progression...38
b) The mitotic spindle: microtubules, centrosomes and genome segregation...39
3.5. Cytokinesis...41
a) Specifying the position of the division plane...42
b) Assembly and constriction of the contractile ring: the cleavage furrow...43
c) Midbody abscission...43
4. Control of the cell cycle...44
4.1. Ubiquitin ligases and cell cycle control...44
4.2. The Anaphase Promoting Complex/Cyclosome (APC/C)...46
a) APC/C architecture and co-activators...46
b) Regulation of APC/C and selective substrate recognition...47
c) Essential functions of APC/C...48
4.3. Quality control of the cell cycle: checkpoints, deregulation and cancer...49
8
MATERIALS & METHODS...57
1. Materials...59
1.1. Buffers and solutions...59
1.2. Oligonucleotides...61
1.3. Primary antibodies...61
1.4. Secondary antibodies...62
1.5. Plasmids...63
1.6. Recombinant proteins...63
1.7. Treatments...64
1.8. Cell lines...64
2. Methods...65
2.1. DNA manipulations...65
a) DNA mutagenesis...65
b) DNA agarose gel electrophoresis...66
c) DNA sequencing and quantification...66
2.2. Cell culture...66
a) Cell maintenance and subculturing...66
b) Freezing / thawing...67
c) Transient transfection...67
Calcium phosphate method...67
Lipofectamine method...67
a) Generation of HeLa DBox stable cell lines...67
b) Cell synchronization...68
Cell synchronization at G1/S transition by a thymidine-aphidicolin double block...68
Cell cycle synchronization at the G1 phase...68
Cell cycle synchronization at the G2 phase...68
Metaphase synchronization...68
Cell cycle synchronization in prometaphase...68
2.3. Animal models...69
2.4. Preparation of cell lysates...70
b) Determination of protein concentration...70
2.5. Immunoprecipitation...70
2.6. Immunoblotting...71
2.7. Immunohistochemistry...71
2.8. Protein stability assays...72
2.9. Protein kinase assays...72
a) GRK2 kinase activity assay...72
b) Phosphorylation of GRK2 by CK2...72
c) Proteomic approach for the identification of phosphorylated peptides of GRK2 by CK2 ...72
2.10. In vitro ubiquitination assays...73
a) Mdm2-GRK2 ubiquitination assay...73
b) APC/C-GRK2 ubiquitination assay...73
2.11. Cell proliferation assays...73
2.12. Cell cycle analysis...73
2.13. Microtubule features analysis...75
a) Measurement of polymerized α-tubulin in vivo...75
b) Microtubule regrowth assays...75
2.14. Alkaline phosphatase staining...75
2.15. Fluorescence microscopy and imaging...76
a) Immunofluorescence...76
b) Image acquisition...76
c) Image quantification...76
d) In vivo time-lapse microscopy...77
2.16. Statistics...78
2.17. Study approval...78
RESULTS...81
1. Mdm2 mediates GRK2 down-modulation in G2 phase but not in mitosis...83
2. The APC/C complex down-modulates GRK2 in mitosis...85
10
2.1. APC/C activity is required to maintain low GRK2 levels during mitosis...86
2.2. APC/C down-modulates GRK2 in a SAC-independent manner...87
3. GRK2 is down-modulated at mitosis in a D-Box-dependent manner...88
3.1. GRK2 D-Box mutation affects protein stability but not Mdm2-mediated regulation or kinase catalytic activity...90
3.2. GRK2 down-modulation in mitosis requires the presence of a D-Box motif and previous phosphorylation at Ser670...91
3.3. The GRK2 D-Box mutant shows an altered interaction with the APC/C complex...93
3.4. A model for the dynamic modulation of GRK2 stability during the cell cycle...94
4. Disruption of GRK2 down-modulation during the cell cycle promotes mitotic defects...95
4.1. HeLa cells stably expressing GRK2 degradation mutants display microtubule aberrations during mitosis...96
4.2. The microtubule anomalies displayed by HeLa cells expressing GRK2 degradation mutants correlate with mitotic spindle defects and increased nuclear irregularity...99
4.3. HeLa cells stably expressing GRK2 degradation mutants undergo changes in mitosis timing...101
5. Potential roles of GRK2 in cytokinesis...102
5.1. GRK2 is localized in the cellular midbody during cytokinesis ...103
5.2. The presence of the GRK2 degradation mutant S670A promotes aberrant cytokinesis...105
5.3. Quantitative and qualitative alterations in the proteome of the midbody in the presence of catalytic or degradation-defective GRK2 mutants...106
6. A role for GRK2 in polyploidy?...111
6.1. Cells stably expressing catalytic- and/or degradation-defective GRK2 mutants exhibit higher rates of polynucleation...111
6.2. GRK2 dosage does not alter per se physiological polyploidy in liver tissue...112
7. Hela cells stably expressing GRK2 degradation mutants undergo changes in the cell cycle timing...113
8. Complex molecular mechanisms prevent GRK2 degradation in G1 phase of the cell cycle...115
8.1. The APC/CCdh1 complex is not able to down-modulate GRK2 in G1 phase...115
8.2. Post-translational modification of the D-Box sequence prevents the degradation of GRK2 protein in G1 phase...116
9. Stemness-like acquired behavior of Hela DBox cells...120
DISCUSSION...125
1. GRK2 protein levels are dynamically regulated throughout the cell cycle...127
1.1. A CDK2-Pin1-Mdm2-APC/CCdc20 axis drives the multilayered degradation pathway of GRK2 in G2 phase and mitosis...127
1.2. CK2 directs the recovery of GRK2 levels in G1 phase...131
2. Functional consequences of GRK2 regulation in the cell cycle...134
2.1. GRK2 down-modulation in G2: centrosome separation...134
2.2. GRK2 down-modulation in mitosis: safeguarding the mitotic spindle...135
a) GRK2 and regulation of spindle inter-centrosomal distance...137
b) Potential consequences of aberrant GRK2 stabilization in chromosome missegregation...138
c) GRK2 and the spindle positioning...139
2.3. GRK2 as a potential regulator of cytokinesis...140
a) GRK2 and midbody functioning and regulation...140
b) Consequences of GRK2-induced cytokinesis abrogation: physiological polyploidy?...143
2.4. Control of GRK2 levels by APC/CCdh1 affects several cellular processes...143
a) Gain-of-function of GRK2 in G1: cell fate determination?...145
3. An integrated view of the regulation and role of GRK2 in the cell cycle...146
CONCLUSIONS...149
CONCLUSIONES...153
REFERENCES...157
ABBREVIATIONS...175
APPENDIX I...181
Supplementary data (digital files)...183
APPENDIX II...185
Abstract
Proteasome-dependent degradation of key kinases represents a major regulatory mecha- nism in the control of cell cycle. Besides being a key player in the desensitization of mani- fold G protein-coupled receptors (GPCRs), the serine/threonine G protein-coupled recep- tor kinase GRK2 is emerging as a signaling node due to its ability to regulate a variety of signaling proteins and cellular functions. Increased GRK2 levels fosters growth factor signaling cascades, proliferation and survival in different types of breast cancer cells. On the other hand, we have reported that timely degradation of GRK2 is part of an intrinsic pathway that ensures cell cycle progression, while kinase stabilization weakens cell cycle arrest promoted by genotoxic drugs. Why and how GRK2 levels change during the differ- ent phases of the cell cycle will help to understand the relevance of this kinase in tumor growth. We find that GRK2 protein levels progressively decay during G2/M as a result of the sequential cooperation of the E3 ligases Mdm2 and APC/C. During G2, the functional interaction of the prolyl-isomerase Pin1 with the CDK2/CyclinA-dependent phosphorylated GRK2 triggers its ubiquitination by Mdm2. Subsequently, at mitosis onset GRK2 decay is promoted by the APC/CCdc20 complex, which recognizes a D-Box destruction motif in GRK2, in a spindle checkpoint-independent manner. Conversely, in the G1 phase several CK2- dependent mechanisms are actively engaged in order to protect GRK2 from the action of APC/CCdh1 complex and functionally upregulate this kinase. Impairment of these timely pro- cesses of GRK2 down-regulation and recovery results in significant alterations in cellular proliferation, G1 duration and mitosis progression. Our data suggest that such aberrations might be related to the reported influence of GRK2 in the EGF-dependent centrosome separation during G2 and with the fact that GRK2 is a key modulator of microtubule dynam- ics through the phosphorylation of the tubulin deacetylase HDAC6. Interestingly, gain-of- function of GRK2 in G1 might help to adapt the dynamics of cell cycle to cell fate decisions, as GRK2-DBox mutant-dependent shortening of G1 correlates with several features of undifferentiating cells.
Moreover, our data unveil the functional interaction of GRK2 with relevant regula- tory factors involved in cytokinesis. GRK2 is specifically located in the midbody and the expression of kinase mutants with impaired stability or activity leads to both alterations in cytokinesis progression and proteomic changes in the midbody composition, thus probably affecting essential processes such as contractile ring organization, cleavage furrow posi- tioning and ingression, or midbody abscission.
Overall, our results suggest that improper regulation of GRK2 in cell cycle might com- promise the fidelity of cell division by means of the rapid progression in G1 and the im- pairment in mitotic spindle assembly and functioning in mitosis as well as in cytokinesis, altogether contributing to polyploidy and chromosomal instability.
Resumen
La degradación proteasomal de quinasas esenciales es el mecanismo de regulación mayori- tario en el control del ciclo celular. Aunque inicialmente caracterizada como uno de los princi- pales efectores de la desensibilización de los receptores acoplados a proteínas G (GPCRs), la quinasa GRK2 se perfila como un nodo emergente en la señalización celular por su capacidad para regular distintas proteínas señalizadoras y procesos celulares. Se ha demostrado que el aumento de los niveles de esta quinasa favorece la señalización en vías de factores de creci- miento y la proliferación y la supervivencia de distintos tipos de células tumorales de mama.
Por otra parte, hemos descrito que fluctuaciones en GRK2 parecen tener un papel regulador de GRK2 en la progresión del ciclo celular, mientras que su estabilización disminuye la eficacia con la que la célula detiene el ciclo en respuesta a sustancias genotóxicas. Por tanto, estudiar cómo y por qué cambian los niveles de GRK2 en las distintas fases del ciclo celular podría ayu- dar a entender el papel de esta quinasa en el crecimiento tumoral. Hemos descubierto que los niveles de GRK2 disminuyen progresivamente durante la fase G2 y mitosis como consecuencia de una cooperación secuencial de las E3 ligasas Mdm2 y APC/C. Durante la fase G2, el com- plejo CDK2/Ciclina A fosforila a GRK2, facilitando la interacción con la prolil-isomerasa Pin1 y permitiendo su posterior ubiquitinación por Mdm2. Por el contrario, el complejo APC/CCdc20 es el responsable de promover la degradación de GRK2 en mitosis, reconociendo un motivo de destrcucción D-Box en su secuencia en un proceso insensible al punto de restricción del huso.
Por otra parte, en la fase G1 varios mecanismos dependientes de la quinasa CK2 protegen a GRK2 de la acción del complejo APC/CCdh1, permitiendo la recuperación de sus niveles. Si se impide que tengan lugar estos procesos secuenciales de disminución y recuperación de GRK2, se producen alteraciones significativas en la proliferación, duración de la fase G1 y progresión de la mitosis. Nuestros datos sugieren que estas alteraciones pueden estar relacionadas con la demostrada influencia de GRK2 en la separación de centrosomas impulsada por EGF en G2 y con el papel de GRK2 en la modulación de la dinámica de microtúbulos mediante la fos- forilación de la tubulin-deacetilasa HDAC6. Resulta interesante que la ganancia de función de GRK2 en G1 parece contribuir a la adaptación de dinámicas de ciclo celular a decisiones de destino celular, ya que el acortamiento de la fase G1 del mutante DBox de GRK2 corresponde con ciertas características de células indiferenciadas.
Además, nuestros datos apuntan a posibles interacciones de GRK2 con importantes reg- uladores del proceso de citoquinesis. Curiosamente, hemos detectado la presencia de GRK2 en el midbody y hemos visto que la expresión de mutantes defectivos en estabilidad o actividad afecta al progreso de la citoquinesis y provoca cambios en el contenido proteico del midbody;
probablemente alterando puntos críticos del proceso como la organización del anillo contráctil, el posicionamiento y hundimiento del surco de división y a la abscisión final del midbody.
En resumen, nuestros resultados sugieren que una degradación inadecuada de GRK2 durante el ciclo celular puede comprometer la fidelidad de la división celular por medio de una rápida progresión en G1 y el deterioro del ensamblaje y la funcionalidad del huso mitótico en la mitosis, así como en la citoquinesis, contribuyendo en conjunto al aumento de poliploidía e inestabilidad genómica.
Introduction
1. GPCRs and GRKs in cellular growth.
Signal transduction networks mediating complex cellular processes, such as proliferation, differentiation, migration or apoptosis, are regulated by growth factors and other messen- gers, which trigger different molecular pathways by activating membrane receptors in either a paracrine or an autocrine manner. Traditionally, the main role in driving cell growth has been ascribed to the activation of tyrosine kinase growth factor receptors (RTKs), which couple ligand binding to downstream signaling cascades and gene transcription (Zwick et al., 2001). However, categorical evidences have also placed the seven-transmembrane domain receptors or G protein-coupled receptors (GPCRs) at the center of the regulation and activation of cellular growth and survival pathways (O’Hayre et al., 2014).
GPCRs comprise the largest family of cell surface receptors involved in signal trans- duction, being activated by a wide variety of external stimuli. Thus, these receptors are crucially involved in almost every physiological process, including neurotransmission, im- mune responses, sensory functions, metabolism, migration or proliferation. Ligands induce conformational changes on the receptors that uncover previously masked heterotrimeric GTP-binding proteins (G protein) binding sites, promoting its activation. Then, G proteins mediate the stimulation of a number of second-messenger systems, small GTPases and an intricate network of kinase cascades, ultimately leading to changes in gene transcrip- tion, cell survival and motility, and normal and malignant cell growth. Many potent mitogens such as lysophosphatidic acid (LPA), thrombin or prostaglandins stimulate cell proliferation through GPCR activation and, accordingly, their aberrant activity or expression contrib- utes to some of the most prevalent human cancers (Gutkind, 1998; Dorsam et al., 2007;
O’Hayre et al., 2014).
Consistent with these essential cellular functions, complex fine-tuning mechanisms regulate GPCRs-mediated signaling. In this regard, desensitization is the main process by which receptor function is transiently switched off even in the presence of agonist stimu- lation. Desensitization is the result of the combination of different mechanisms including receptor-G-protein uncoupling as a consequence of receptor phosphorylation and the in- ternalization from the cell surface. Second messenger–dependent protein kinases such as PKA and PKC can phosphorylate GPCRs in specific consensus sequences, leading to G protein uncoupling. Moreover, agonist-occupied GPCR are specifically phosphorylated by G protein-coupled kinases (GRKs), what in turn promotes binding of cytoplasmic β-arrestin molecules to the receptors, sterically inhibiting further interactions with G proteins, and eventually resulting in a rapid attenuation of G protein-mediated receptor responsiveness and transient receptor internalization (reviewed in Ferguson, 2001; Ramachandran et al., 2012; Smith et al., 2016) (Figure I1).
Introduction
24
The GRK family contains seven serine-threonine kinases (GRK1-GRK7), with a com- mon involvement in GPCR desensitization but with several differences in domain structure, expression patterns and physiological functions. Based on its homology, GRKs can be di- vided into three classes: GRK1-like or visual GRKs (GRK1/Rhodopsin kinase and GRK7,) primary found in retina; GRK2-like or β-Adrenergic receptor kinase family (β-ARK) (GRK2 and GRK3), and GRK4-like (GRK4, GRK5 and GRK6). GRK4 is predominantly located in testes whereas the rest of the non-visual kinases are expressed ubiquitously throughout the body (Premont & Gainetdinov, 2007) (Figure I2).
Arrestin AP2
Clathrin-coated pit G protein
Membrane reinsertion α
β γ
P P P α β γ GTP GDP GRK
Early endosome Sorting endosome
Lysosomal degradation Recycling vesicle
Dephosphorylation Activation
Agonist
Ramachandran et al., 2012 Figure I1. Mechanisms of GPCRs desensitization and internalization. GRKs phos- phorylate GPCRs allowing β-arrestin recruitment and G-protein uncoupling. These intracel- lular events lead to receptor desensitization, endocytosis and, depending on the receptor, preferential recycling to the plasma membrane or lysosomal degradation.
As shown in Figure I2, all GRK isoforms include a specific amino-terminal domain, displaying homology to RGS proteins (thus termed RH domain); which could regulate GPCR signaling by phosphorylation-independent mechanisms allowing for instance spe- cific GRK2/3 interaction with Gαq/11 proteins (Carman et al., 1999). A central catalytic or kinase domain belonging to the AGC kinase family is present in all GRKs, whereas a more variable carboxyl-terminal domain is important for the localization and translocation of ki- nases to the membrane by means of either post-translational modifications or sites of inter- action with lipids or membrane proteins (Homan & Tesmer, 2014; Nogués et al., 2017b). In addition, the members of the GRK4-like subfamily contain a functional nuclear localization signal (NLS), and specifically GRK5 and GRK6 have been shown to translocate to the nucleus. Overall, these multi-domain characteristics could contribute to endow GRKs with a wide range of functional features (Penela et al., 2010; Watari et al., 2014).
Beyond its canonical role in GPCRs desensitization, GRKs are involved in GPCR- mediated G protein-independent signaling through β-arrestins, since the latter are scaffold proteins for many signaling partners, thus promoting additional signaling pathways from GPCR signalosomes (Shenoy and Lefkowitz, 2011) (Figure I3). It has been suggested that both the ligand-induced conformational changes and the phosphorylation pattern (called phosphorylation barcoding) imprinted by GRKs in GPCRs could be important for determin- ing the balance between G protein activation, GPCRs desensitization, and GRK/β-arrestin
5 5 4 5
8 1 7
1
1 513 547 665 689
PH Domain AGC
C-Tail RH
1 19 181 454 511 531 558
RH domain Kinase Domain RH
αN C-TailAGC
0 8 1 6
1 1
RH domain Kinase Domain
450 507 527 574
palmitoylation R
H AGC C-Tail
RH domain Kinase Domain
Cys-COOC 3H S
GRK1/GRK7
GRK2/GRK3
GRK4/GRK5/GRK6 39
44
39
βARKct
C T
9 8 6 5
9 4
αN
αN
N-terminal Central core C-terminal
RGS domain Catalytic domain Membrane binding GPCR binding domain GRK1-like
subfamily
GRK2-like subfamily
GRK4-like subfamily
Adapted from Homan & Tesmer, 2014 Figure I2. Structure of the GRKs family. Common and specific domain features of the three subfamilies of GRKs. The involvement of given domains in localization and protein interactions is indicated.
Introduction
26
mediated signaling (Liggett et al., 2011). However, the detailed mechanism underlying GRKs specificity for given GPCRs or their relative ability to promote GPCR desensitiza- tion versus G protein-independent signaling remain unclear. On the other hand, it is worth noting that the impact of changes in GRK expression and functionality in cellular functions might also involve non-GPCR targets. Thus, GRKs are emerging as signal transducers by themselves (Figure I3), as a result of functional or scaffolding interactions with a wide variety of substrates. GRKs have been reported to interact with (and in many cases phos- phorylate) a variety of non-GPCR proteins (Penela et al., 2003; Ribas et al., 2007; Penela et al., 2010b; Gurevich et al., 2012; Watari et al. 2014; Nogués et al., 2017b). GRK partners include components of cell networks related to inflammation, cell motility, proliferation and, interestingly, cell cycle, in the case of GRK2 (Penela et al., 2010a, Nogués et al., 2017a; b) and GRK5 (Michal et al., 2011).
2. GRK2 as a key node in signal transduction pathways related to the hallmarks of cancer.
Although initially identified as a key player in the desensitization and internalization of man- ifold GPCRs, the ubiquitous and essential GRK isoform GRK2 has been shown to interact with and/or phosphorylate a variety of non-GPCR membrane receptors, such as receptor tyrosine kinases, and other downstream effectors of signal transduction cascades. Such complex network of GRK2 connections participates in the modulation of key aspects of cell proliferation, survival, motility or metabolic homeostasis. Besides, the expression and func- tion of this kinase is finely modulated by a variety of mechanisms and altered GRK2 levels have been noted in different tumor contexts (Nogués et al., 2017a).
Adapted from Premont & Gainetdinov, 2007 Figure I3. GRK-dependent signaling. In addition to its canonical role in triggering GPCR phosphorylation and β-arrestin binding, GRKs display their own signalosome as a result of functional or scaffolding interactions with a wide variety of cellular partners.
2.1. The complex GRK2 interactome: implications in cell metabolism, motility, proliferation and survival.
An increasing number of studies have reported the ability of GRK2 to phosphorylate a wide range of non-GPCR membrane receptors (as PDGF or EGF receptor tyrosine kinases) and other downstream effectors of different signaling cascades such as Smads, HDAC6, ezrin, IRS1, phosducins or p38 MAPK among others. There are also evidences of GRK2 acting as a scaffold protein, establishing dynamic interactions with several partners as for instance Gαq, PI3K/Akt, GIT1, MEK, IRS1, EPAC, Pin1, or Mdm2 (reviewed in Penela et al., 2010b; Evron et al., 2012; Gurevich et al., 2012; Penela et al., 2014; Nogués et al., 2017a).
This complex canonical and non-canonical GRK2 interactome would underlie the role of this protein in different cellular processes and physiological functions (Figure I4).
PAWR/PAR4 Dynamin 2
Not determined Positive effect Negative effect
Consequences of GRK2/partner associations APC
PDEg GRB2
Src
Cdk2 Pin1
Cell proliferation
Cell migration
Cell survival Cell metabolism
RKIP
Insulin R PAF receptor
Ezrin Glut4
TLR2,3,4,7 Gαq
AKT1
Phosphorylation
Caveolin 1
PI3Kg PI3Ka AKT1
Tubulin
ERK1/2
RGS2 RGS4
Radixin GIT1
MEK1
p38
HDAC6
Hsp90
Smad2/3 GRK2
S1P1 EGFR PDGFR KCNJ3
Metabolic
GPCR DRD2
Chemokine Receptors
Adapted from Mayor Jr. et al., 2016
Figure I4. GRK2 interactome. Combinations of sequential or parallel functional interac- tions of GRK2 with a variety of GPCR and other signal transduction partners underlie the role of this protein in cellular processes such as cell proliferation, migration, survival, or metabolism.
Introduction
28
A growing number of reports indicate that GRK2 plays an important integrative role in the homeostasis of cellular metabolism, energy production and expenditure (Ciccarelli et al., 2012; Vila-Bedmar et al., 2012; 2015; Lucas et al., 2015; Mayor et al., 2017). In addi- tion to modulating the actions of several GPCR related to the control of metabolism, such as adrenergic receptors, enhanced GRK2 levels have been shown to markedly impair insulin sensitivity in vivo through signaling inhibition at the IRS1 level (Cipolletta et al., 2009; Garcia-Guerra et al., 2010), or by inhibition of insulin-mediated glucose transport by interfering with Gq/11 function (Usui et al., 2005) or insulin-stimulated glycogen synthesis (Shahid et al., 2007). Moreover, it has been demonstrated that decreasing GRK2 levels or activity prevents or reverts key aspects of the obese and diabetic phenotype triggered by high-fat diet: impedes further body weight gain, normalizes glucose intolerance and leads to preserved insulin sensitivity in skeletal muscle and in liver, thus maintaining glucose homeostasis (Anis et al., 2004; Garcia-Guerra et al., 2010; Vila-Bedmar et al., 2015; Sato et al., 2015). In addition, it has been described that GRK2 can localize to the mitochondrial outer membrane by means of the cellular stress–induced MAPK-mediated modification of the protein and the interaction with the chaperone Hsp90. The consequences of such mito- chondrial translocation are controversial because both detrimental (increased cytochrome C release and apoptosis) and protective (increased biogenesis and ATP production) effects have been reported (Fusco et al., 2012; Huang et al., 2014). Taken together, these data put forward GRK2 as a potential molecular hub linking aging, insulin resistance, obesity, and cardiovascular diseases, and point to this protein as a candidate for therapeutic strategies for obesity or type 2 diabetes-related diseases.
Several evidences indicate a relevant role of GRK2 in cellular motility. GRK2 ap- pears to modulate different steps of the chemotactic process such as receptor sensing, cell polarization, membrane protrusion or adhesion in response to different stimuli in a cell type-specific way (Penela et al., 2014). Besides regulating manifold chemokine and other GPCR involved in cell migration, GRK2 phosphorylates ezrin (Cant et al., 2005) and radixin (Kahsai et al., 2010), both ERM proteins involved in local F-actin polymerization-dependent membrane protrusion. Also, GRK2 can transiently interact with the GIT1 scaffold protein, thus potentiating the activation of the Rac1/PAK/MEK/ERK pathway in response to adhe- sion and S1P stimulation, leading to F-actin cortical remodeling, enhanced focal adhesion turnover and increased epithelial cell migration (Penela et al., 2008; Penela et al., 2014).
Regarding the tubulin cytoskeleton, besides the intriguing fact that GRK2 phosphorylates beta-tubulin, with unknown functional implications (Pitcher et al., 1998), GRK2 has been reported to control microtubule (MT) dynamics through the activating phosphorylation of the cytosolic histone deacetylase type II protein HDAC6, what eventually leads to in- creased alpha-tubulin deacetylation. This modification takes place at the cell leading edge and is necessary for the positive effect of HDAC6 in the fibronectin or EGF-induced migra- tion of epithelial cells and fibroblasts (Lafarga et al., 2012). Interestingly, phosphorylation
of GRK2 itself by other kinases appears to determine the preferential partner or substrate in the cell migration process (reviewed in Penela et al, 2014). GRK2 phosphorylation in tyrosine residues enhances the interaction with GIT-1, whereas MAPK-mediated modifica- tion would inhibit interaction with GIT1 and GPCR, whereas allowing HDAC6 phosphoryla- tion. Therefore, changes in the subcellular localization, kinase activity and phosphorylation status of GRK2 would allow dynamic and stimuli-specific signaling with all these relevant partners affecting cell migration, allowing its sequential and coordinated participation in several steps of the motility process.
On the other hand, GRK2 also modulates cell proliferation and mitogenic signaling through the regulation of MAPK pathways at different levels, in a cell-type and stimuli- dependent manner. Contrary to its canonical negative role in G protein-dependent GPCR signaling, GRK2 can potentiate MAPK activation and growth signaling in response to S1P and integrins (Penela et al., 2008), EGF (Wan et al., 2003), the Smoothened receptor (Meloni et al., 2006) or CXCR7 (Lipfert et al., 2013) in specific cell types. The mitogenic modulatory effect of GRK2 would also be based on its ability to phosphorylate or dynami- cally interact with important modulators/effectors engaged along the MAPK pathway, such as GIT1, MEK1, RhoA, Epac, PDEγ, RKIP or Pin1 upon stimulation by EGF or several mitogenic GPCRs (Wan et al., 2003; Penela et al., 2008; Eijkelkamp et al., 2010; Penela et al. 2010b; Deiss et al., 2012; Robinson & Pitcher, 2013; Nogués et al., 2017a; b). In addition, we and others have reported a role for GRK2 in ensuring a proper and timely progression of G1/S and G2/M transitions of the cell cycle in response to extrinsic and intrinsic signals, respectively (reviewed in Penela et al. 2010a). In particular, GRK2 levels are down-regulated during the G2/M transition and this transient degradation is required for normal cell cycle progression. Interestingly, the default GRK2 protein decay in G2 is prevented in the presence of DNA damaging agents that trigger cell cycle arrest such as doxorubicin. Actually, such accumulation of GKR2 inversely correlated with the activation of p53 triggered by G2/M checkpoints mechanisms, helping to restrict the apoptotic face of arrested cells (Penela et al., 2010a). Accordingly, we hypothesized that GRK2 might allow cells to undertake genotoxic stress by protecting against cell cycle arrest and potentiating survival response.
In this regard, GRK2 is also able to interact with some key players in several process- es related to cellular stress response and growth arrest, such as p38, Smad2/3, PI3K, AKT or Hsp90 (reviewed in Penela et al., 2010b; Nogués et al., 2017a; b), what might positively impact on cell survival and resisting cell death. For instance, GRK2 phosphorylates p38, a critical player in apoptosis or survival in a cell-type specific context and a mediator of p53 activation in response to different stresses (Bulavin & Fornace, 2004), impairing the binding of the p38 upstream activator MKK6 and thus inhibiting p38-dependent signaling (Peregrin et al., 2006). GRK2 also interacts with both PI3K and AKT proteins, although the functional
Introduction
30
outcome of such interactions is not straightforward, as positive and negative effects have been described in a cell type-specific context. GRK2 contributes to the GPCR induced AKT activation by means of its association with the catalytic subunit of PI3Kγ, while a direct interaction with the regulatory subunit p85 facilitates AKT activation in the context of cell cycle of epithelial cells (Rivas et al., unpublished results), whereas in non-epithelial cells a GRK2-mediated inhibition of Akt phosphorylation and canonical activation has been shown (Liu et al., 2005).
2.2. Regulation of the activity and protein turnover of GRK2.
The abundance and activation status of a particular GRK in a given cellular context, would directly affects receptor signaling and desensitization and their interactions with additional partners. Consistent with the complexity of its interactome, GRK2 expression levels, ac- tivity and subcellular location are tightly regulated. Our group has focused over the past decade on the study of the different mechanisms of GRK2 modulation, particularly of those related to the control of GRK2 degradation.
At the level of kinase activity, the GRK2 RH domain serves as an intra-molecular scaf- fold that maintains the small lobe of the kinase domain in a state that is competent to phos- phorylate without requiring previous phosphorylations on its activation loop for full activity (Homan & Tesmer, 2014), which can be triggered upon interaction with activated GPCR and Gβγ subunits. GRK2 can also interact with different inhibitors such as calcium-binding proteins, α-actinin, Hsp90 or caveolin. In addition, this kinase can also undergo posttrans-α-actinin, Hsp90 or caveolin. In addition, this kinase can also undergo posttrans-actinin, Hsp90 or caveolin. In addition, this kinase can also undergo posttrans- lational modifications that can regulate its catalytic activity, drive the kinase towards differ- ent substrates or modulate its subcellular location or proteolysis rate (reviewed in Ribas et al., 2007; Penela et al., 2010b; Penela, 2016). For instance, S-nitrosylation of Cys residues within its catalytic domain inhibit GRK2 kinase activity towards GPCRs (Whalen et al., 2007). On the other hand, phosphorylation of GRK2 by PKA, PKC or c-Src has been shown to enhance its membrane localization and/or catalytic activity whereas phosphorylation by ERK1/2 at Ser670 decreases it (Sarnago et al., 1999; Elorza et al., 2000; Penela et al., 2003). Interestingly, as mentioned above this latter modification at the C-terminus of GRK2 promotes a switch in its substrate specificity, fostering the acquisition of a distinctive com- petent conformation at the active site that allows, for instance, phosphorylation of HDAC6 (Lafarga et al., 2012). Since allosteric communication has been suggested to take place between the C-tail PH domain and the RH domain of GRK2, it is possible that structural alterations caused by covalent modifications at the C-terminus could be transmitted to the catalytic domain, affecting its conformation.
Regarding expression levels, non-transcriptional mechanisms are actively involved in the regulation of GRKs, particularly of GRK2 (reviewed in Penela, 2016: Nogués et al., 2017a). In fact, little is known about the regulation of GRK2 at the transcription or mRNA
translation level. In vascular cells, some studies have revealed that its transcriptional activ- ity is increased by phorphol esters or activation of Gαq or α1-adrenergic signaling pathways whereas proinfl ammatory cytokines promote the opposite effect (Ramos et al., 2000; Pene- proinflammatory cytokines promote the opposite effect (Ramos et al., 2000; Pene- la et al., 2003).
The control of GRK2 protein stability appears to play a major role in determining the cellular dosage of this kinase. The main degradation pathways involved in protein turn- over include the lysosomal, caspase, calpain and ubiquitin-dependent proteasome sys- tems. In this regard, reports have shown that degradation of GRK2 is mostly mediated by calpains or the proteasome upon GPCR activation. In this context, scaffold proteins and phosphorylation by different kinases play an important role in modulating GRK2 stability (Figure I5). Both beta-arrestin-dependent c-Src and MAPK phosphorylation of GRK2 drive GPCR-induced degradation of the protein independently of each other in a proteasome- dependent manner (Penela et al., 1998; Penela et al., 2001; Elorza et al., 2003). Interest- ingly, our group identified Mdm2 as the main E3 ligase implicated in GRK2 turnover by the proteasome pathway (Salcedo et al., 2006), for what a previous phosphorylation of GRK2 at Ser670 by MAP kinases was needed whereas tyrosine phosphorylation was dispens- able (Nogués et al., 2011). This latter modification is critical instead for binding to Hakai, a novel RING-finger type E3 ubiquitin-ligase (Moñino S. and Penela P., unpublished obser- vations). This ligase related to the regulator of receptor tyrosine kinases Cbl, possesses a unique phosphotyrosine-binding domain which consensus of interaction (consisting in pTyr residues surrounded by acidic amino acids) is found in GRK2 when it is tyrosine- phosphorylated in response to PDGF receptor stimulation. It is unknown however if this ligase can be also recruited to GRK2 in the context of GPCR activation. Adding more lay- ers of complexity, it is also worth noting that Mdm2 phosphorylation by Akt in response to stimulation of growth factor receptors such as IGFR leads to E3 ligase translocation to the nucleus, resulting in enhanced GRK2 stability and expression levels (Salcedo et al., 2006).
Interaction of GRK2 with the heat shock protein Hsp90 also helps in the maintenance of GRK2 levels, by contributing to the proper folding and maturation of the newly synthesized GRK2 (Luo & Benovic, 2003).
Notably, the phosphorylation status also affects GRK2 stability in a cell cycle context, thus governing the fluctuations in kinase levels taking place during cell cycle progression, as we mentioned before. Phosphorylation of GRK2 at Ser670 by CDK2-Cyclin A and the subsequent binding of the prolyl-isomerase Pin1, are required for transient GRK2 protea- somal degradation during the G2/M transition (Penela et al., 2010), although the E3 ligase involved was not identified.
Introduction
32
c-Src Tyr13/86/92 S670
Ub Ub
UbUb
GRK2
GRK2
GRK2 Mdm2
ERK1/2 PI3K
Hakai?
GRK2
GRK2GRK2 GRK2
Pin1?
Akt
GPCR
Mdm2
S G2
M
GRK2 CDK2 S670
GRK2 Pin1 Mdm2 E3?
Adapted from Nogués, 2014 & Penela, 2016
IGF-1R
Figure I5. Regulation of GRK2 degradation in different contexts. GPCR activation trig- gers the sequential recruitment of GRK2, β-arrestin and c-Src, leading to GRK2 phosphor- ylation on tyrosine residues and promoting its degradation by the proteasome pathway.
Receptor activation also promotes β-arrestin-mediated association of Mdm2 to MAPK- phosphorylated GRK2 at Ser670, resulting in ubiquitination at defined residues and kinase proteolysis. On the other hand, there is a tumoral deregulation of GRK2 turnover, in which IGF-1 stimulation induces GRK2 protein accumulation in a PI3K dependent manner, by relieving the Mdm2-dependent degradation of GRK2. In the cell cycle context, GRK2 is degraded in a GPCR-independent way, in which the adaptor role of β-arrestins is dispens- able. GRK2 phosphorylation on Ser670 by CDK2-CyclinA results in GRK2 interaction with the prolyl-isomerase Pin1, leading to degradation at G2/M transition. Whether GPCR-de- pendent MAPK phosphorylation of GRK2 at Ser670 creates a conditional phosphodegron for the E3-RING ligase Mdm2 involving the action of Pin1 as in the cell cycle is not known, nor the E3 ligases responsible for GRK2 degradation during cell cycle progression have been identified.
2.3. GRK2 in cancer progression.
Importantly, alterations in GRK2 expression and kinase activity, previously reported to take place in pathological conditions such as inflammation or cardiovascular disease, are also starting to be noted in several tumor contexts (reviewed in Nogués et al., 2017a; b). Can- cer is caused by sequential genetic and epigenetic alterations that disrupt the regulatory circuits governing normal cell proliferation and homeostasis. This multistep pathologic pro- cess involves the progressive occurrence of molecular modifications and cellular adapta- tions by selective pressure over many years, which leads to considerable heterogeneity and variability among tumors. However, most tumors share essential alterations in cell physiology, which together promote malignant transformation and are termed the hallmarks of cancer (Hanahan and Weinberg, 2011).
Regulatory molecular nodes able to integrate multiple upstream inputs and trigger diverse downstream outputs are particularly suitable to act as non-oncogenic contribu- tors to malignant transformation and progression. In this context, GRK2 is emerging as a potentially relevant oncomodulator given its functional connections with the most relevant signaling networks required for the proper function, homeostasis, and viability of the cell (Figure I6).
Nogués et al., 2017
Figure I6. GRK2 as a potential modulator of the hallmarks of cancer. The ability to regulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to
Introduction
34
In a previous section, we have summarized the functional connections of GRK2 with pathways modulating cell motility, proliferation, survival or cell metabolism. Regarding the later, important metabolic adaptations take place during cancer progression as a result of fluctuations in oxygen or nutrient availability, altered mitochondrial function and redox status, modulation of autophagy and proteostasis or modifications in proliferation or in the microenvironment (Lehuédé et al., 2016). It also has been suggested that patients with in- sulin resistance/obesity have a higher risk of developing several types of cancer (Klil-Drori et al., 2016). Given the roles of GRK2 in modulating cell metabolism networks in the con- text of obesity and diabetes, it is tempting to suggest that changes in GRK2 levels/activity taking place in tumor cells may modulate metabolic networks or mitochondrial functions.
The noted role of GRK2 in cell motility may also contribute to cancer development and progression, since a number of GRK2 partners are important players in tumor cell invasion, including plasma membrane GPCRs (for ligands as S1P), integrins, or EGF re- ceptors, as well as cytoskeleton modulators such as RhoA, Rac1, or ERM proteins. Other potential GRK2 targets, such as the chemokine receptors CXCR4 and CXCR7, are highly expressed in a range of tumors and play a role in metastasis, although their role in cancer progression is not fully understood (O’Hayre et al., 2014). The implications of GRK2 in the regulation of cell proliferation and survival pathways detailed above also suggest a power- ful link between the kinase and tumorigenesis. Besides the already mentioned pathways, GRK2 phosphorylates Smad2/3 upon stimulation of the ALK5 receptor by TGFβ, inhibit-β, inhibit-, inhibit- ing Smad complex nuclear shuttling and thus hindering pro-apoptotic TGFβ effects and enhancing its tumor-promoting role (Ho et al., 2005, 2007). Also in this regard, it has been recently reported that GRK2 potentiates Hedgehog/smoothened-mediated transformation in fibroblasts (Zhao et al., 2016).
GRK2 levels could also play a relevant role in the modulation of signaling in other cell types present in the tumor microenvironment, such as the tumor-associated vasculature or infiltrated immune cells. GRKs are known to control chemokine-triggered signaling in lymphocytes and neutrophils during inflammation, although the specific impact of changes in GRK2 dosage in immune cells during cancer progression has not been investigated (Nogués et al., 2017a; b). On the other hand, tumor microvasculature is usually highly angiogenic and leaky, displaying enlarged and dilated vessels lined with immature walls due to the loss of pericytes, leading to deficient blood supply. Subsequent hypoxia prompts secretion of different pro-angiogenic and pro-inflammatory factors, perpetuating aberrant vascularization and inflammation and thus facilitating cancer progression (Potente et al., 2011). In this context, GRK2 is able to integrate several pathways involved in endothelial cell activation and maturation, such as those mediated by S1P, VEGF, PDGF-BB, and TGFβ1 receptors. Decreased GRK2 levels take place in endothelial cells in breast tumors, leading to altered balance of TGFβ signaling, which controls both the activation and resolu-β signaling, which controls both the activation and resolu-signaling, which controls both the activation and resolu-
tion phases of angiogenesis via the timely modulation of the opposite effects of ALK1 and ALK5 receptors (Rivas et al., 2013, 2014). We have reported that GRK2 loss impedes en- dothelial cells differentiation and fusion into tubular structures and hampers the recruitment of pericytes, leading to immature and leaky vessels and enhanced recruitment of tumor- associated macrophages, thus fostering the growth of tumors in mice (Rivas et al., 2013).
Interestingly, recent data from our laboratory indicate a relevant oncomodulator role for GRK2 in breast tumorigenesis (Nogués et al., 2016). GRK2 upregulation emerges as a convergent feature of the stimulation of diverse pathways altered in luminal breast cancer, in parallel to that of other key proteins such as HDAC6 or Pin1, known to be overexpressed in these tumoral contexts (Lee et al., 2008; Zhou and Lu, 2016). In such situations, as well as in settings of increased phosphorylation of GRK2 on S670 by means of hyperactivation of ERK1/2 (often found in both luminal and basal breast cancer contexts), the switch-on of the GRK2-HDAC6-Pin1 signaling module would help to perpetuate cell proliferation and increased survival (Nogués et al., 2016; 2017a).
The changes and roles of GRK2 in different tumors may be different. GRK2 is over- expressed in pancreatic cancer and might serve as potential indicator of unfavorable prog- nosis (Zhou et al., 2016). On the other hand, GRK2 appears to play an inhibitory role in IGF1-induced hepatocellular carcinoma proliferation and migration, and a GRK2-specific peptide inhibitor increased tumor mass upon xenograft transplantation of HEK293 cells (Fu et al., 2013). Down-modulation of GRK2 has been reported in Kaposi sarcoma (Hu et al., 2015). A more precise understanding of the mechanisms underlying such tumor- and cell type–specific regulation of GRK2 levels and functionality and of its impact in signaling net- works and cellular functions will help to gain further insight on its integrated role in cancer development and to design future therapeutic strategies (Nogués et al., 2017a ;b).
In this Thesis we have focused our interest in the implications of GRK2 in cell cycle progression. Our group had reported that transient changes in GRK2 levels and its phos- phorylation status at Ser670 modulate the G2/M transition in a receptor-independent man- ner, putting forward an unforeseen role for GRK2 in cell growth control. It is tempting to suggest that CDK2-triggered GRK2 down-regulation would allow cell cycle progression in normal conditions by preventing untimely functional interactions of GRK2 with other partners (Penela et al., 2010a). The mechanisms involved in the tight regulation of GRK2 protein levels during cell cycle, the consequences of deregulating these fluctuations, and the potential roles of this kinase, especially during mitosis, are the key aspects addressed during this thesis.
Introduction
36
3. The cell cycle progression.
Simply stated, the cell cycle couples rounds of DNA replication with chromosomal segre- gation, guaranteeing the proper distribution of the duplicated material into two daughter cells. This highly ordered and tightly regulated process functions in a unidirectional and irreversible manner and can be divided into four classical and differentiated phases (Nor- bury and Nurse, 1992). It is well stablished that the DNA synthesis or replication phase (S phase) and segregation phase (Mitosis) are separated by two gap phases, named G1 and G2 (Figure I7). Those proteins responsible for ensuring appropriate progression through the cell division are called cyclins, which form heterodimers with cyclin-dependent kinases (CDKs) thus phosphorylating a wide range of cellular proteins to promote G1 phase entry and progression, to boost DNA synthesis during S phase and to allow the cell to prepare during G2 to trigger chromosome segregation during mitosis. Interestingly, additional non- canonical functions of these proteins have already been reported (Hydbring et al., 2016).
Moreover, additional controls or checkpoints exist in the division process to ensure an orderly sequence of events (Heartwell and Weinert, 1989).
Prophase Prometaphase
Metaphase
Mitosis Interphase M
G1
G2 S
Anaphase
Telophase Cytokinesis
G0Quiescence
Centrosome MTs
Nucleus
Membrane DNA
DNA replication Centrosome duplication
Centrosome maturation and separation
NEBD Chromatin condensation Chromosomes Centrosome KT
separation
Astral MTs Interpolar MTs
Capture of MTs by KTs Bipolarity establishment
Spindle assembly MT-KT attachment + chromosome bi-orientation =
SAC inactivation Metaphase plate
Chromosome segregation
Spindle elongation Cleavage furrow and nuclear envelope
formation Spindle midzone
Midbody Abscission
Figure I7. The cell cycle. Graphical summary of the main processes that are carried out in each phase during cell cycle progression.
3.1. G1 phase.
The G1 phase constitutes the gap between mitosis and DNA synthesis, and together with the G2 phase comprises the interphase, the growth period of the cell cycle. During this phase, cell initiates the synthesis of mRNA and proteins required for DNA replication before entry intro S phase. This process is controlled and limited by growth factors, the extracel- lular matrix and cell-cell contacts as well as stress conditions, thus upon deprivation of essential factors, cells become quiescent and enter into G0 (Agami & Bernards, 2002).
In favorable conditions, sequential activation of distinct CDK-cyclin complexes leads to stepwise activation of signaling pathways and to the “switch-on” of transcription factors responsible for S phase programing, such as E2F or c-myc, among others, and inactivation of cell cycle repressors by means of phosphorylation-mediated engagement of ubiquitin- dependent proteasome degradation. Thus, cyclin D-dependent kinases (Cdk4 and Cdk6) are activated in response to mitogenic signaling and phosphorylate Rb to partially relieve the repression of E2F-dependent gene expression. Among the transcriptional targets of E2F there are additional G1 cyclins that form complexes with a CDK (cyclin E-Cdk2), which fully activate G1/S transcription through a positive feedback loop that allows passage of the restriction point. Finally, Cdk2 also phosphorylates and inactivates APC/CCdh1, which in combination with the inhibitor binding Emi1 removes the last barrier to S-phase entry (Ver- meulen et al., 2003; Robert, 2015; Sherr & Bartek, 2017) (see also sections 4.2 and 4.3).
3.2. S phase.
Once all requirements and transcription factors “get ready” during G1, G1/S transition checkpoints are disabled and the cell starts to replicate its DNA content, as a prelude to its segregation to the daughter cells at mitosis. In addition to DNA, also centrosomes are duplicated during this phase (as detailed below in the mitotic spindle section). This replica- tion is achieved by between 103 and 105 pairs of replication forks initiating at sites scattered throughout the genome. S phase events are tightly regulated to ensure complete chromo- somal DNA replication while avoiding more than one replication (Hutchison, 1995).
3.3. G2 phase.
G2 phase is the other gap, between DNA synthesis and mitosis, where cells prepare for division. Although other essential processes such as centrosome maturation and, in most cases, separation take place then, the main objective of this phase is ensuring DNA in- tegrity before chromosome segregation. For this purpose, the cell counts on DNA repair mechanisms and a potent DNA checkpoint mechanism able to promote cell cycle arrest in the presence of DNA damage until it gets repaired (Vermeulen et al., 2003; Robert, 2015).
Compelling evidence point to CDK1 Tyr phosphorylation as the main regulatory mechanism that ensures the coordination between cell growth and cell division. Thus, transition of G2
Introduction
38
phase into mitosis is actively prevented by the Wee1-mediated phosphorylation of the cy- clin-dependent kinase (CDK) Cdk1 on Tyr15, while its de-phosphorylation by Cdc25 leads to CDK1 activation and mitosis onset. In addition, the kinase Greatwell helps progression of G2 into mitosis by suppressing protein phosphatase 2A (PP2A)-B55 that counteracts cyclin B-Cdk1, though its mammal orthologous Mastl is only required for mitosis progres- sion. Moreover, two pathways acting upstream of Wee1 and Cdc25, the mitogen-activated protein kinases stress-nutritional response and the cell geometry sensing pathways are involved in the regulation of CDK1/cyclin B to guarantee that the cell divides with the proper size and nutrient state (Wood & Nurse, 2015).
3.4. Mitosis.
During mitosis, chromosomes and cytoplasm are divided into two daughter cells. This phase is characterized by significant alterations in cell shape, cytoskeleton reorganization and chromatin structure. A precise regulation of all mitotic events is crucial for proper chro- mosome segregation (Pinheiro & Sunkel, 2012; Robert, 2015).
a) Mitotic progression.
In animal cells, mitosis can be subdivided into six phases, characterized by clearly distinc- tive features (Figure I7) (Pinheiro & Sunkel, 2012; Robert, 2015).
• Prophase. Centrosomes finish to separate to opposite sides of the nucleus, locat- ing the position of the future mitotic spindle poles, and serving as MT organization centers (MTOCs) in which MTs start to organize. Chromosomes condense pro- gressively in sister chromatids attached by centromeres. Complete DNA conden- sation and nuclear envelope breakdown determine the end of prophase.
• Prometaphase. As will be detailed in sections below, the nuclear envelope break- down (NEBD) allows MTs to interact with and guide chromosomes towards the equatorial area of the mitotic spindle in a process mediated by motor proteins.
Mitotic spindle assembly takes place during this phase through the polymeriza- tion and attachment of astral-MTs around centrosomes and the cell cortex, and interpolar-MTs in the central area and the chromosomes kinetochore.
• Metaphase. Chromosomes align at the spindle equatorial area forming the meta- phase plate. Correct alignment allows Spindle Assembly Checkpoint (SAC) ful- fillment in order to enable spindle tension and chromosome segregation, or cell cycle arrest to avoid missegregation defects.
• Anaphase. Once all chromosomes are correctly aligned and SAC is disabled, co- hesin deactivation and separase activation by the Anaphase Promoting Complex or Cyclosome (APC/C) permit sister chromatids separation, pulled to opposite poles of the cell via elongation of the spindle.
• Telophase. Nuclear envelope reassembles around the separated chromosomes while spindle disassembles. The formation of the central spindle or spindle mid- zone initiates, resulting in the formation of the cleavage furrow.
• Cytokinesis. A contractile ring forms and constricts around the midbody until ab- scission allows final partitioning of the cytoplasm and the physical separation of the two daughter cells. This step will be further detailed in a section below.
b) The mitotic spindle: microtubules, centrosomes and genome segregation.
The mitotic spindle is a bipolar dynamic macromolecular structure mainly constructed from MTs and regulatory proteins such as MT-associated or motor proteins, and is responsible for ensuring proper chromosome segregation into the two daughter cells during mitosis.
Mitotic spindle assembly is essential for maintaining genome integrity, since its defects have been shown to lead to chromosome misalignment and eventually aneuploidies (re- viewed in Potapova and Gorbsky, 2017). This organized process depends mainly on the tightly regulated de novo formation of MTs or MT nucleation, via centrosome-, chromatin- and microtubule- mediated pathways that contribute to mitotic spindle assembly in a both redundant and collaborative manner (reviewed in Prosser and Pelletier, 2017).
The centrosome, consisting in two centrioles surrounded by a pericentriolar protein matrix, is the main MT-organizing center (MTOC) in most animal cells, identified at the center of the astral MT arrays that define the spindle poles and contribute to spindle as- sembly (Bettencourt-Dias & Glover, 2007). Every centriole must duplicate once in every cell cycle (cell cycle control) and each new centriole must form next to every pre-existing centriole (copy number control), in order to maintain a constant number of centrioles in proliferating cells (Figure I8). At a morphological level, the control of centrosome duplica- tion involves the formation and dissolution of two different types of connections between centrioles, a protein linker named “S-M linker” and the ‘G1–G2 tether’. S-M linker is formed between the mother and daughter centrioles at late in S phase and is a key structure for the prevention of centriole re-duplication. This connection is dissolved by the action of PLK1 and separase in a process known as “centrosome disengagement” which takes place at M/G1 transition. This step licenses centrosome for duplication in the next S phase of the cell cycle under the control of PLK4 activity. It is proposed that timely activation of PLK4 might lead to inactivation of the SCF–FBXW5 E3-ubiquitin ligase and accumulation of the ligase substrate SAS-6, which is a main organizing component of the procentriole structure
Introduction
40
(Nigg & Stearns, 2011). However, the molecular details of procentriole formation remains to be understood. The second linker, the G1-G2 tether is formed following centriole disen- gagement in G1, connecting the proximal ends of the two parental centrioles from G1 to late G2 (reviewed in Agircan et al., 2014). This linker involves several proteins critical for its function such as C‑Nap1, rootletin or CEP68, among others, and ensures microtubule nucleation from a single microtubule-organizing center. During G2, this linker disassembles by the centrosome disjunction process mediated by the NIMA-related kinase Nek2, which phosphorylates C-Nap1 and rootelin to allow centrosomes to separate in a kinesin (Eg5)- mediated manner and form the two poles, thus supporting assembly of the mitotic spindle (Faragher & Fry, 2003). Interestingly, recent studies have demonstrated the implication of GRK2 in the timely regulation of this process by means of the direct phosphorylation of the kinase Mst2 (an upstream activator of Nek2) in a PLK1-independent manner in response to EGF receptor stimulation during the early course of G2 (So et al., 2013).
M
G2
S
G1
Cohesin ring
Separase
CDK1Plk1 PCM
C-Nap1
G1-G2 tether: Rootletin, Cep68 (i) Centriole
disengagement
APC/C SCF-FBXW5 SAS6
Plk4
(ii) Centriole duplication
initiation S-M linker
SCFβ-TrCP Plk4
(iii) Procentriole elongation (iv) Centrosome
maturation and separation
P
(v) Bipolar spindle assembly
Centrobin
α/β-tubulin Procentriole structure Astrin
AurAPlk1 CDK1
PCM Microtubule
C-Nap1 Nek2A
Daughter centriole Chromosome
Mother centriole
Cell cycle
Adapted from Wang et al., 2014
Figure I8. The centrosome cycle. Graphical summary of the main processes that drive centrosome cycle during the cell cycle (see text for details).
On the other hand, spindle MTs are joined head-to-head α/β-tubulin dimers that form a growing polar structure through the addition of components to its plus ends and emanating from γ-tubulin rings (Kollman et al., 2011), resulting in an antiparallel bipolar MT array. MT dynamic stability allows rapid remodeling of the interphase cytoskeleton to form the mitotic spindle through continuous, rapid cycles of polymerization and depolymerization, grow- ing and shrinking the structure before ultimately disassembling (Mitchison & Kirschner, 1984). The mitotic spindle is comprised by different types of MTs. Astral MTs originate from spindle poles and interact with the cell cortex, thus being essential for spindle positioning.
Interpolar MTs also arise from spindle poles but grow towards the opposite pole in order to separate them and provide stability to the spindle. Most interpolar MTs attach to the chro- mosomes kinetochore (KT), building K-fibers that mediate chromosome movement, and are termed KT-MTs. The consequent chromosome alignment is crucial for mitotic progres- sion since the SAC is not deactivated until all kinetochores have established a proper and stable attachment to the spindle.
Furthermore, it is important to note that all spindle MTs are formed by the same α/β- tubulin heterodimers, thus highlighting the necessity of a fine-tuned control of specific spin- dle MTs for guiding chromosomes to equatorial locations during mitosis. In this regard, recent evidence have revealed the existence of a “tubulin code”, in which tubulin post- translational modifications such as acetylation or detyrosination, account for subcellular differentiation of distinct MT populations to perform different functions in order to coordinate spindle assembly and KT-based chromosome motility during mitosis (Barisic & Maiato, 2016).
3.5. Cytokinesis.
Cytokinesis is the final step of mitosis, in which the content of a single cell is partitioned into two, following chromosomes segregation. This process is initiated with the assembly of an equatorial contractile ring through cortical remodeling coordinated by the anaphase spindle. Then, this ring contracts ingressing a cytokinetic furrow and forming a midbody that directs an abscission to separate the plasma membrane (Figure I9). Cytokinesis failure has been shown to result in genetically instable tetraploid cells with extra centrosomes that will perturb chromosome segregation in subsequent cell divisions, eventually contributing to cancinogenesis (Ganem et al., 2007), thus underlying the importance of a tight regula- tion in time and space of this process.