RESUMEN
Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni-NTA pull-down, and detecting protein-protein interaction and substrate Y-phosphorylation. This protocol is adaptable for studies examining the function of other extracellular kinases. For complete details on the use and execution of this protocol, please refer to Backe et al. (2023)1 and Sánchez-Pozo et al. (2018).2.
Asunto(s)
Proteínas Tirosina Quinasas , Familia-src Quinasas , Humanos , Familia-src Quinasas/metabolismo , Fosforilación , Proteína Tirosina Quinasa CSK/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismoRESUMEN
Secreted, or extracellular, heat shock protein 90 (eHsp90) is considered a recent discovery in eukaryotes. Over the last two decades, studies have provided significant supporting evidence that implicates eHsp90 both in normal cellular processes such as wound healing and in the development of human pathologies and diseases including fibrosis and cancer. In the early 2000s, Eustace et al. demonstrated that eHsp90 promotes the invasion of breast cancer cells by binding to and regulating the activity of an extracellular matrix (ECM) remodeling enzyme, the matrix metalloproteinase 2 or MMP2. Interestingly, inside mammalian cells, Hsp90 is an essential chaperone that interacts with hundreds of newly synthesized proteins, known as "clients," that require Hsp90's assistance to perform their function. Several methods are routinely used to characterize the role and impact of Hsp90 on a client protein's functionality in vitro and in vivo. However, the mechanistic role of eHsp90 is less well-defined since, so far, only a handful of extracellular client proteins have been identified. Here, we describe methods to characterize the impact of the secreted chaperone on MMP2 activity, the most characterized extracellular client of eHsp90. The procedures described here can be applied and adapted to characterize other extracellular clients, particularly members of the MMP family.
Asunto(s)
Metaloproteinasa 2 de la Matriz , Neoplasias , Animales , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Chaperonas Moleculares , Proteínas HSP90 de Choque Térmico/metabolismo , Matriz Extracelular/metabolismo , Mamíferos/metabolismoRESUMEN
c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.
Asunto(s)
Proteínas Tirosina Quinasas , Secretoma , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Fosfotransferasas , Fosforilación , Dominios Homologos src , Comunicación Celular , Familia-src QuinasasRESUMEN
The molecular chaperone Heat Shock Protein-90 (Hsp90) is known to interact with over 300 client proteins as well as regulatory factors (eg. nucleotide and proteins) that facilitate execution of its role as a chaperone and, ultimately, client protein activation. Hsp90 associates transiently with these molecular modulators during an eventful chaperone cycle, resulting in acquisition of flexible structural conformations, perfectly customized to the needs of each one of its client proteins. Due to the plethora and diverse nature of proteins it supports, the Hsp90 chaperone machinery is critical for normal cellular function particularly in response to stress. In diseases such as cancer, the Hsp90 chaperone machinery is hijacked for processes which encompass many of the hallmarks of cancer, including cell growth, survival, immune response evasion, migration, invasion, and angiogenesis. Elevated levels of extracellular Hsp90 (eHsp90) enhance tumorigenesis and the potential for metastasis. eHsp90 has been considered one of the new targets in the development of anti-cancer drugs as there are various stages of cancer progression where eHsp90 function could be targeted. Our limited understanding of the regulation of the eHsp90 chaperone machinery is a major drawback for designing successful Hsp90-targeted therapies, and more research is still warranted.
RESUMEN
The striated body wall muscles of Caenorhabditis elegans are a simple model for sarcomere assembly. Previously, we observed deletion mutants for two formin genes, fhod-1 and cyk-1, develop thin muscles with abnormal dense bodies (the sarcomere Z-line analogs). However, this work left in question whether these formins work in a muscle cell autonomous manner, particularly since cyk-1(∆) deletion has pleiotropic effects on development. Using a fast acting temperature-sensitive cyk-1(ts) mutant, we show here that neither postembryonic loss nor acute loss of CYK-1 during embryonic sarcomerogenesis cause lasting muscle defects. Furthermore, mosaic expression of CYK-1 in cyk-1(∆) mutants is unable to rescue muscle defects in a cell autonomous manner, suggesting muscle phenotypes caused by cyk-1(∆) are likely indirect. Conversely, mosaic expression of FHOD-1 in fhod-1(Δ) mutants promotes muscle cell growth and proper dense body organization in a muscle cell autonomous manner. As we observe no effect of loss of any other formin on muscle development, we conclude FHOD-1 is the only worm formin that directly promotes striated muscle development, and the effects on formin loss in C. elegans are surprisingly modest compared to other systems.
Asunto(s)
Caenorhabditis elegans/patogenicidad , Proteínas Fetales/metabolismo , Forminas/metabolismo , Músculo Estriado/metabolismo , AnimalesRESUMEN
Epithelial cell-cell junctions have dual roles of accommodating morphological changes in an epithelium, while maintaining cohesion during those changes. An abundance of junction proteins has been identified, but many details on how intercellular junctions respond to morphological changes remain unclear. In Caenorhabditis elegans, the spermatheca is an epithelial sac that repeatedly dilates and constricts to allow ovulation. It is thought that the junctions between spermatheca epithelial cells undergo reversible partial unzipping to allow rapid dilation. Previously, we found that EXC-6, a C. elegans protein homolog of the human disease-associated formin INF2, is expressed in the spermatheca and promotes oocyte entry. We show here that EXC-6 localizes toward the apical aspect of the spermatheca epithelial junctions, and that the EXC-6-labeled junction domains "unzip" and dramatically flatten with oocyte entry into the spermatheca. We demonstrate that the C-terminal tail of EXC-6 is necessary and sufficient for junction localization. Moreover, expression of the tail alone worsens ovulation defects, suggesting this region not only mediates EXC-6 localization, but also interacts with other components important for junction remodeling.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Epiteliales/metabolismo , Animales , Polaridad Celular , Humanos , Uniones IntercelularesRESUMEN
The cytoskeleton is the basic machinery that drives many morphogenetic events. Elongation of the C. elegans embryo from a spheroid into a long, thin larva initially results from actomyosin contractility, mainly in the lateral epidermal seam cells, while the corresponding dorsal and ventral epidermal cells play a more passive role. This is followed by a later elongation phase involving muscle contraction. Early elongation is mediated by parallel genetic pathways involving LET-502/Rho kinase and MEL-11/MYPT myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase in another. While the LET-502/MEL-11 pathway appears to act primarily in the lateral epidermis, here we show that FEM-2 can mediate early elongation when expressed in the dorsal and ventral epidermis. We also investigated the early elongation function of FHOD-1, a member of the formin family of actin nucleators and bundlers. Previous work showed that FHOD-1 acts in the LET-502/MEL-11 branch of the early elongation pathway as well as in muscle for sarcomere organization. Consistent with this, we found that lateral epidermal cell-specific expression of FHOD-1 is sufficient for elongation, and FHOD-1 effects on elongation appear to be independent of its role in muscle. Also, we found that fhod-1 encodes long and short isoforms that differ in the presence of a predicted coiled-coil domain. Based on tissue-specific expression constructions and an isoform-specific CRISPR allele, the two FHOD-1 isoforms show partially specialized epidermal or muscle function. Although fhod-1 shows only impenetrant elongation phenotypes, we were unable to detect redundancy with other C. elegans formin genes.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microfilamentos/genética , Morfogénesis/genética , Fosfoproteínas Fosfatasas/genética , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Epidermis/embriología , Epidermis/metabolismo , Forminas , Especificidad de Órganos/genética , FenotipoRESUMEN
Formins are regulators of actin filament dynamics. We demonstrate here that two formins, FHOD-1 and EXC-6, are important in the nematode Caenorhabditis elegans for ovulation, during which actomyosin contractions push a maturing oocyte from the gonad arm into a distensible bag-like organ, the spermatheca. EXC-6, a homolog of the disease-associated mammalian formin INF2, is highly expressed in the spermatheca, where it localizes to cell-cell junctions and to circumferential actin filament bundles. Loss of EXC-6 does not noticeably affect the organization the actin filament bundles, and causes only a very modest increase in the population of junction-associated actin filaments. Despite absence of a strong cytoskeletal phenotype, approximately half of ovulations in exc-6 mutants exhibit extreme defects, including failure of the oocyte to enter the spermatheca, or breakage of the oocyte as the distal spermatheca entrance constricts during ovulation. Loss of FHOD-1 alone has little effect, and we cannot detect FHOD-1 in the spermatheca. However, combined loss of these formins in double fhod-1;exc-6 mutants results in profound ovulation defects, with significant slowing of the entry of oocytes into the spermatheca, and failure of nearly 80% of ovulations. We suggest that EXC-6 plays a role directly in the spermatheca, perhaps by modulating the ability of the spermatheca wall to rapidly accommodate an incoming oocyte, while FHOD-1 may play an indirect role relating to its known importance in the growth and function of the egg-laying muscles. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Microfilamentos/metabolismo , Oocitos/metabolismo , Ovulación/metabolismo , Citoesqueleto de Actina/inmunología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Femenino , Forminas , Masculino , Proteínas de Microfilamentos/genética , Ovulación/genéticaRESUMEN
eIF4E plays a conserved role in initiating protein synthesis, but with multiple eIF4E isoforms present in many organisms, these proteins also adopt specialized functions. Previous RNAi studies showed that ife-3, encoding the sole canonical eIF4E isoform of Caenorhabditis elegans, is essential for viability. Using ife-3 gene mutations, we show here that it is maternal ife-3 function that is essential for embryogenesis, but ife-3 null progeny of heterozygous animals are viable. We find that zygotic ife-3 function promotes body growth and regulates germline development in hermaphrodite worms. Specifically, the normal transition from spermatogenesis to oogenesis in the hermaphrodite germline fails in ife-3 mutants. This failure to switch is reversed by inhibiting expression of the key masculinizing gene, fem-3, suggesting ife-3 resembles a growing number of genes that promote the sperm/oocyte switch by acting genetically as upstream inhibitors of fem-3.
RESUMEN
Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line-associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.