In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells

Humphrey, David M.; Rajfur, Zenon; Vázquez, Martha; Scheswohl, Danielle M; Schaller, Michael D.; Jacobson, Ken; Imperiali, Barbara

doi:10.1074/jbc.m502726200

Cited by 31 publications

(17 citation statements)

References 53 publications

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“…Importantly for cellular applications, the photo-byproduct of NPE-caged species, o-nitrosoacetophenone, has previously been shown to be cellularly inert (Nguyen et al 2004;Humphrey et al 2005). This byproduct is less reactive than the analogous o-nitrosobenzaldehyde produced by irradiation of widely used ortho-nitrobenzyl-derived caging groups (Kaplan et al 1978).…”

Section: Uncaging Of Cpy31pax (4c)mentioning

confidence: 99%

“…As a complement to these approaches, the synthesis of caged phosphopeptides, which enable the controlled release of specific phosphorylated species upon photolysis, was introduced (Rothman et al 2002). Recently, a general method for the synthesis of these probes has facilitated the application of caged phosphopeptides in cellular studies (Nguyen et al 2004;Humphrey et al 2005). Expanding on this work, and as part of an initiative to develop generalizable approaches for the preparation of full-length caged phosphoproteins, we report the semisynthesis and biochemical characterization of a mutant of the 61-kDa protein paxillin that includes a caged phosphotyrosine (cpTyr) residue at position 31 within the N terminus (Fig.…”

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confidence: 99%

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Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Vogel

Imperiali

2007

Protein Science

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Caged phosphopeptides and phosphoproteins are valuable tools for dissecting the dynamic role of phosphorylation in complex signaling networks with temporal and spatial control. Demonstrating the broad scope of phosphoamino acid caging for studying signaling events, we report here the semisynthesis of a photolabile precursor to the cellular migration protein paxillin, which is a complex, multidomain phosphoprotein. This semisynthetic construct provides a powerful probe for investigating the influence that phosphorylation of paxillin at a single site has on cellular migration. The 61-kDa paxillin construct was assembled using native chemical ligation to install a caged phosphotyrosine residue at position 31 of the 557-residue protein, and the probe includes all other binding and localization determinants in the paxillin macromolecule, which are essential for creating a native environment to investigate phosphorylation. Following semisynthesis, paxillin variants were characterized through detailed biochemical analyses and by quantitative uncaging studies.

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Section: Uncaging Of Cpy31pax (4c)mentioning

confidence: 99%

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confidence: 99%

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Vogel

Imperiali

2007

Protein Science

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“…To further investigate the role of phosphorylated FAK in live cells, a peptide corresponding to a region of FAK incorporating a caged autophosphorylation site that is sensitive to photoactivation was microinjected into live cells. These studies indicate a direct role for FAK autophosphorylation in regulating forward protrusion of lamellipodia at the leading edge of cells migrating on 2D surfaces [47]. In another study, using GFP as a CALI reagent, irradiation of GFP-a-actinin resulted in detachment of stress fibres from focal adhesions, whereas inactivation of GFP-FAK did not, suggesting that, FAK is not essential for physical anchorage of focal adhesions to the actin cytoskeleton [48].…”

Section: Signallingmentioning

confidence: 96%

“…Molecular mutagenesis of natural fluorescent proteins such as green fluorescent protein (GFP) has produced a variety of fluorescent proteins with distinct spectral characteristics [11,12]. Advances in material sciences, has also resulted in [35,37]; FAK [49]; FAK, Src, Paxillin [45]; Calpain and Talin [52] Quantum dots EGFr [107]; Migration/invasion [15,16] Photoactivation FAK [47]; Integrin trafficking [37] FRET/FLIM Src [24]; FAK [38]; Calpain [25] CALI FAK [48] FRAP b3 integrin [35]; FAK [108] TIRFM R-Ras [109]; Actin, Microtubules and focal adhesions [65,66]. the generation of new classes of synthetic fluorophores such as Quantum dots; inorganic nanocrystals composed of a semi-conductor core and ZnS shell that possess discrete fluorescent emission properties [13].…”

Section: New Applications Of Fluorescent Reporter Technologymentioning

confidence: 98%

Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover

Carragher

2008

Clin Exp Metastasis

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Recent advances in microscopic imaging technology, fluorescent reporter reagents, 3-dimensional (3D) cell models and multiparametric image analysis have enhanced our ability to model and understand complex cell physiology. Extension of these approaches to live cell, kinetic studies allows further spatial and temporal understanding of a multitude of dynamic functional events, including tumour cell invasion. Recent in vivo and 3D in vitro studies reveal how tumour cells utilize a diverse variety of mechanisms to permit invasion through 3D tissue environments. Such high degrees of diversity and plasticity between invasion mechanisms present a significant challenge to the successful treatment of malignant cancer. This review examines how advances in time-resolved imaging has contributed to the characterization of distinct modes of invasion and their associated molecular mechanisms. Specifically, we highlight the development of fluorescent reporter molecules and their incorporation into more predictive 3D in vitro and in vivo models, to enhance mechanistic analysis of tumour invasion. We also highlight the latest advances in kinetic imaging instrumentation applicable to in vitro and in vivo models of tumour invasion. We discuss how multiparametric image analysis can be used to interpret image data generated by these approaches. We further discuss how these approaches can be integrated into drug discovery pipelines to facilitate evaluation and selection of candidate drugs and novel pharmaceutical compositions, targeting multiple invasive mechanisms.

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“…However there is the possibility to perform functional studies using caged molecules. For example Humphrey et al [99] revealed the importance of Focal Adhesion Kinase (FAK) in lamellopodia extension, by photoactivating a non-functional, truncated FAK that competed with the endogenous protein for effector binding. Clearly the possibility of performing similar studies in vivo is an exciting prospect.…”

Section: Trackingmentioning

confidence: 99%

Lymphocyte tracking and interactions in secondary lymphoid organs

et al. 2007

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The induction of an adaptive immune response is an essential step in the generation of long-lasting, protective immunity to pathogens. Many studies over the last few decades have identified the cell populations involved in the generation of antigen-specific immunity and elucidated the role of many important molecules. However, because of the low precursor frequency of antigen-specific cells, the immune system must be highly dynamic, surveying most sites of the body. Recent studies have, therefore, begun to examine how the cells of the immune system interact in vivo during the induction of an immune response, identifying new and important roles for certain molecules and revealing how previously unrecognised alterations in cell-cell interactions can have significant implications for the resulting immune response. Here we review some of these recent studies that provide a valuable insight into the mechanisms involved in the induction of immunity.

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In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells

Cited by 31 publications

References 53 publications

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover

Lymphocyte tracking and interactions in secondary lymphoid organs

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