Vanesa Lafarga

The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

GRK2 is a ubiquitous member of the G protein‐coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein‐coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist‐regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non‐GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context‐specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2.

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α‐tubulin deacetylase. We identify G protein‐coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α‐tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α‐tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2‐K220R or GRK2‐S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6‐mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.

A new p38 MAP kinase-regulated transcriptional coactivator that stimulates p53-dependent apoptosis

The p38 mitogen‐activated protein kinase (MAPK) signaling pathway plays an important role in stress‐induced cell‐fate decisions by orchestrating responses that go from cell‐cycle arrest to apoptosis. We have identified a new p38 MAPK‐regulated protein that we named p18Hamlet, which becomes stabilized and accumulates in response to certain genotoxic stresses such as UV or cisplatin treatment. Overexpression of p18Hamlet is sufficient to induce apoptosis, whereas its downregulation reduces the apoptotic response to these DNA damage‐inducing agents. We show that p18Hamlet interacts with p53 and stimulates the transcription of several proapoptotic p53 target genes such as PUMA and NOXA. This correlates with enhanced p18Hamlet‐induced recruitment of p53 to the promoters. In proliferating cells, low steady‐state levels of p18Hamlet are probably maintained by a p53‐dependent negative feedback loop. Therefore, p18Hamlet is a new cell‐fate regulator that links the p38 MAPK and p53 pathways and contributes to the establishment of p53‐regulated stress responses.

Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in muscle differentiation.

The chromatin‐remodelling complex SNF2‐related CBP activator protein (SRCAP) regulates chromatin structure in yeast by modulating the exchange of histone H2A for the H2A.Z variant. Here, we have investigated the contribution of H2A.Z‐mediated chromatin remodelling to mammalian cell differentiation reprogramming. We show that the SRCAP subunit named ZNHIT1 or p18Hamlet, which is a substrate of p38 MAPK, is recruited to the myogenin promoter at the onset of muscle differentiation, in a p38 MAPK‐dependent manner. We also show that p18Hamlet is required for H2A.Z accumulation into this genomic region and for subsequent muscle gene transcriptional activation. Accordingly, downregulation of several subunits or the SRCAP complex impairs muscle gene expression. These results identify SRCAP/H2A.Z‐mediated chromatin remodelling as a key early event in muscle differentiation‐specific gene expression. We also propose a mechanism by which p38 MAPK‐mediated signals are converted into chromatin structural changes, thereby facilitating transcriptional activation during mammalian cell differentiation.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML

Chemotherapy-resistant acute myeloid leukemia may respond to inhibition of ATR or ATM. New hope for AML patients A pair of papers provides new hope for patients with acute myeloid leukemia (AML) by showing that the DNA replication checkpoint pathway is a viable target for therapeutic intervention. By integrating survival data from 198 treated AML patients with gene expression data for genes encoding proteins involved in the regulation of DNA replication, David et al. identified the CHEK1 gene and its product, the DNA replication checkpoint kinase CHK1, as both a prognostic indicator of survival and a therapeutic target to overcome resistance to the current standard of chemotherapy. The patients had all received standard-of-care chemotherapy. Patients with high expression of CHEK1 in their AML cells had reduced survival, and AML patient cells with high CHK1 abundance were resistant to the toxic effects of the DNA replication inhibitor cytarabine. CHK1 is activated by the kinase ATR in response to DNA replication stress arising from DNA damage. The identification of CHEK1 expression as high in lymphomas and leukemias, including AML, prompted Morgado-Palacin et al. to investigate targeting ATR and ATM, the most upstream kinases in the DNA damage response, as possible AML therapies. AML cells with oncogenic rearrangements in MLL are particularly resistant to genotoxic therapies that form the backbone of AML treatment. Inhibiting ATR resulted in death of AMLMLL cells in culture and exhibited antitumoral activity in AMLMLL mouse models. Inhibiting ATM also prolonged survival of the allograft mouse model, indicating that targeting the DNA damage response pathways alone or in combination with other chemotherapeutic agents may be beneficial in patients with AML. Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AMLMLL cells (with an MLL-ENL fusion and a constitutively active N-RAS) independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AMLMLL and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AMLMLL mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Roles of GRK2 in Cell Signaling Beyond GPCR Desensitization: GRK2-HDAC6 Interaction Modulates Cell Spreading and Motility

GRK2 modulates tubulin acetylation dynamics in an HDAC6-dependent manner to affect epithelial cell spreading and motility. G protein–coupled receptor kinase 2 (GRK2) is a ubiquitous, essential protein kinase that is emerging as an integrative node in many signaling networks. Moreover, changes in GRK2 abundance and activity have been identified in several inflammatory, cardiovascular disease, and tumor contexts, suggesting that those alterations may contribute to the initiation or development of pathologies. GRKs were initially identified as key players in the desensitization and internalization of multiple G protein–coupled receptors (GPCRs), but GRK2 also phosphorylates several non-GPCR substrates and dynamically associates with a variety of proteins related to signal transduction. Ongoing research in our laboratory is aimed at understanding how specific GRK2 interactomes are orchestrated in a stimulus-, context-, or cell type–specific manner. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6) that modulates cell spreading and motility. HDAC6 is a major cytoplasmic a-tubulin deacetylase that is involved in cell motility and adhesion. GRK2 dynamically and directly associates with and phosphorylates HDAC6 to stimulate its a-tubulin deacetylase activity at specific cellular localizations, such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. GRK2-HDAC6–mediated regulation of tubulin acetylation also modulates cellular spreading. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to epithelial cell migration.

Efficacy of ATR inhibitors as single agents in Ewing sarcoma.

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.

The interplay between G protein-coupled receptor kinase 2 (GRK2) and histone deacetylase 6 (HDAC6) at the crossroads of epithelial cell motility

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative node in cell migration control. In addition to its canonical role in the desensitization of G protein-coupled receptors involved in chemotaxis, novel recently identified GRK2 substrates and interacting partners appear to mediate the GRK2-dependent modulation of diverse molecular processes involved in motility, such as gradient sensing, cell polarity or cytoskeletal reorganization. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6), a major cytoplasmic α-tubulin deacetylase involved in cell motility and adhesion. GRK2 dynamically associates with and phosphorylates HDAC6 to stimulate its α-tubulin deacetylase activity at specific cellular localizations such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to aberrant epithelial cell migration.

p18Hamlet Mediates Different p53-Dependent Responses to DNA Damage Inducing Agents

Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38α (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38α as well as the activation of specific target genes by p53. We recently characterized a new p38α substrate, named p18Hamlet (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18Hamlet protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that Hamlet can also mediate the cell cycle arrest induced in response to γ-irradiation, by participating in the p53-dependent up-regulation of the cell cycle inhibitor p21Cip1 (CDKN1A).

The SMN Tudor SIM-like domain is key to SmD1 and coilin interactions and to Cajal body biogenesis

ABSTRACT Cajal bodies (CBs) are nuclear organelles involved in the maturation of spliceosomal small nuclear ribonucleoproteins (snRNPs). They concentrate coilin, snRNPs and the survival motor neuron protein (SMN). Dysfunction of CB assembly occurs in spinal muscular atrophy (SMA). Here, we demonstrate that SMN is a SUMO1 target that has a small ubiquitin-related modifier (SUMO)-interacting motif (SIM)-like motif in the Tudor domain. The expression of SIM-like mutant constructs abolishes the interaction of SMN with the spliceosomal SmD1 (also known as SNRPD1), severely decreases SMN–coilin interaction and prevents CB assembly. Accordingly, the SMN SIM-like-mediated interactions are important for CB biogenesis and their dysfunction can be involved in SMA pathophysiology.