RESUMO
ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.
Assuntos
Proteínas Ativadoras de GTPase/genética , Hipertensão/genética , Doenças Pulmonares Intersticiais/genética , Animais , Criança , Feminino , Homozigoto , Humanos , Leucocitose/genética , Leucocitose/imunologia , Doenças Pulmonares Intersticiais/patologia , Linfocitose/genética , Linfocitose/imunologia , Masculino , Camundongos , Linhagem , Sequenciamento do Exoma , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Humanos , Fosforilação , Ligação Proteica , Proteína Quinase C/genética , Proteômica/métodos , Transdução de Sinais , Especificidade por SubstratoRESUMO
The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.
Assuntos
Movimento Celular/fisiologia , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Animais , Humanos , Camundongos , Fosforilação , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Integrin-mediated cell adhesion is essential for cell migration, mechanotransduction and tissue integrity. In vivo, these processes are regulated by complex physicochemical signals from the extracellular matrix (ECM). These nuanced cues, including molecular composition, rigidity and topology, call for sophisticated systems to faithfully explore cell behaviour. Here, we discuss recent methodological advances in cell-ECM adhesion research and compile a toolbox of techniques that we expect to shape this field in future. We outline methodological breakthroughs facilitating the transition from rigid 2D substrates to more complex and dynamic 3D systems, as well as advances in super-resolution imaging for an in-depth understanding of adhesion nanostructure. Selected methods are exemplified with relevant biological findings to underscore their applicability in cell adhesion research. We expect this new "toolbox" of methods will allow for a closer approximation of in vitro experimental setups to in vivo conditions, providing deeper insights into physiological and pathophysiological processes associated with cell-ECM adhesion.
Assuntos
Adesão Celular , Movimento Celular , Matriz Extracelular , Movimento Celular/fisiologia , Humanos , Matriz Extracelular/metabolismo , Animais , Integrinas/metabolismoRESUMO
Here, we describe the synthesis and biological characterization of 32 novel phenylalanine and leucine dipeptides modified on both the N and C termini by salicylic acid and aromatic or alicyclic amines, respectively. All compounds displayed antiproliferative activity in the tested cancer cell lines and eight of the compounds exhibited single digit micromolar GI50 values. Treated cells rapidly detached from surface of tissue culture dishes and we found that focal adhesion kinase (FAK), p130CAS and paxillin, which are important regulators of cell adhesion, were dephosphorylated at Y397, Y410 and Y118, respectively. The most potent compound reduced proliferation in the HCT-116 cell line in a dose-dependent manner, as shown by a decrease in 5-bromo-2'-deoxyuridine incorporation into DNA. Furthermore, this compound increased the levels of several apoptotic markers, including activated caspases, and increased site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.
Assuntos
Antineoplásicos/farmacologia , Dipeptídeos/farmacologia , Leucina/farmacologia , Fenilalanina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/síntese química , Dipeptídeos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Leucina/síntese química , Leucina/química , Estrutura Molecular , Fenilalanina/síntese química , Fenilalanina/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The ability of cancer cells to adopt various migration modes (the plasticity of cancer cell invasiveness) is a substantive obstacle in the treatment of metastasis, yet still an incompletely understood process. We performed a comparison of publicly available transcriptomic datasets from various cell types undergoing a switch between the mesenchymal and amoeboid migration modes. Strikingly, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was one of three genes that were found upregulated in all amoeboid cells analyzed. Accordingly, downregulation of MALAT1 in predominantly amoeboid cell lines A375m2 and A2058 resulted in decrease of active RhoA (Ras homolog family member A) and was accompanied by the amoeboid-mesenchymal transition in A375m2 cells. Moreover, MALAT1 downregulation in amoeboid cells led to increased cell proliferation. Our work is the first to address the role of MALAT1 in MAT/AMT (mesenchymal to amoeboid transition/amoeboid to mesenchymal transition) and suggests that increased MALAT1 expression is a common feature of amoeboid cells.
RESUMO
Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Quinase C/metabolismo , Animais , Movimento Celular , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Fosfotreonina/metabolismo , Podossomos/metabolismo , Ligação Proteica , Pseudópodes/metabolismo , Fibras de Estresse/metabolismo , Quinases da Família src/metabolismoRESUMO
CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.