Conceived and designed the experiments: Jd SL CJ RR. Performed the experiments: SL CJ RR. Analyzed the data: Jd SL CJ FN RR. Contributed reagents/materials/analysis tools: RR. Wrote the paper: Jd SL CJ AH. Other: Is PI on the grant employing Simone Lemeer: MS.
The authors have declared that no competing interests exist.
Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other.
We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles.
Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways
PTKs are essential regulators of virtually all biological processes and they have pivotal roles in development and disease
To date, kinase activity profiling was done using peptide arrays spotted onto glass coverslips. These are incubated with a kinase (mixture) and radioactively labeled ATP, [γ-33P]ATP. Detection of phosphorylation is done by autoradiography, using phosphoimaging technology for quantification
We have used the PTK activity profiling described above for purified PTKs. Moreover, we determined PTK activity profiles in complex mixtures of zebrafish embryo lysates. The zebrafish is increasingly being used as a model for human disease
Here, we report that peptide chip arrays allowed us to reproducibly determine PTK activity profiles in 1 day old zebrafish embryos. Knock down of the Src family kinases (SFKs), Fyn and Yes, led to specific changes in the PTK activity profile. Moreover, knock down of the non-canonical Wnt11 that induced similar phenotypes as the Fyn and Yes knock downs
Here, we have used peptide array technology for the first time in zebrafish embryos to determine PTK activity profiles. The peptide arrays consist of 144 peptides, each 15 amino acids in length covering known phosphorylation sites (
(a) Fluorescence of the panel of 144 peptides is shown after 60 min (endpoint). The fluorescently labeled control peptides at the periphery indicate the grid of 144 different peptides. (b) Kinetics of EFS peptide phosphorylation by control embryo lysate (pink), Fyn/Yes morpholino injected embryo lysate (green) and Fyn/Yes morpholino, Fyn/Yes RNA co-injected embryos (Fyn/Yes rescue, brown) is shown with a data point every three min and the fluorescence in arbitrary units. (c,d) Endpoint measurements of peptide phosphorylation by Nacre- or Fyn/Yes morpholino-injected embryo lysates. (e–h) Quantification of PTK profiles. The relative intensities of peptide phosphorylation by lysates of control, non-injected embryos were plotted against Nacre- or Fyn/Yes knock down embryos. The fluorescently labeled reference spots all lie within the indicated oval. (g,h) are a close-up of the low range of panels e and f. The five highlighted peptides are: 1,EFS; 2, FAK2; 3, LTK; 4, PAXI and 5, SYN1.
Next, we determined PTK activity profiles following morpholino-mediated knock down of the Src family kinases (SFKs), Fyn and Yes. Previously, we have shown that Fyn/Yes knock downs affect convergence and extension (CE) cell movements
(g–l) Relative intensities of phosphorylation of a selection of five peptides. The signal was determined in duplicate and calculated relative to non-injected wild type control (set to 100%). (m–n) Relative intensities of phosphorylation of the same set of five peptides by purified Yes and Fyn kinases.
To ensure that the Fyn/Yes knock down-induced differences in PTK profile were specific, Fyn/Yes morpholinos and synthetic RNA encoding Fyn and Yes were co-injected at the one-cell stage. Consistent with our previous results, these co-injections effectively rescued the Fyn/Yes knock down phenotype (
Previously, we have shown that Fyn and Yes operate in a pathway that converges with non-canonical Wnt signalling to regulate convergent extension cell movements during vertebrate gastrulation (
Peptide phosphorylation of Fyn/Yes (FYmorph), Wnt11 (Wntmorph) or Nacre knock down was determined relative to wild type (WT), which was set to 100%. The average of a duplicate experiment is depicted here. Phosphorylation intensities were color coded, <25%, red; 25–75%, yellow and 75–125%, green. Protein identities of the peptides are indicated as well as their start and end residues. The sequence of the peptides and numerical representation of the data is given in
Here we show that it is feasible to produce effective PTK activity profiles of zebrafish embryos by utilising peptide chip technology. Moreover, we demonstrate that the system is amenable to functional assays. We detected significant, reproducible changes in PTK activity profiles from morpholino knock down embryos that were partially rescued by co-injection of the corresponding mRNAs. This technology allows for the rapid identification of candidate proteins/signaling pathways associated with specific genes.
Zebrafish were kept and the embryos were staged as described before
Embryos (24 hpf) were deyolked with deyolking buffer (½ Ginzburg Fish Ringer) without calcium
Micro-array experiments were performed using PamChip peptide arrays run on a PamStation4 instrument (PamGene, 's Hertogenbosch, the Netherlands). Four temperature controled peptide chips were run in parallel by pumping the sample up and down through the 3-dimensional porous chip. Data was captured by real-time imaging of the fluorescence signal by CCD imaging
The positions of the peptides on the chip are indicated as well as their protein names, the amino acid residue numbers and the sequences of the peptides. Fluorescence intensities for all peptides were determined quantitatively and expressed as arbitrary units. Relative intensities were calculated within individual peptide chips to allow for comparison between different experiments. Averages of two independent experiments were determined and are expressed here as relative signal compared to the signal in wild type, non-injected control embryos (set to 100%). Absolute levels of fluorescence are indicated for wild type, non-injected control (arbitrary units). Signals below the detection threshold level are indicated by a -. The fluorescence intensities of the PTK activity profiles of purified Fyn and Yes are depicted in arbitrary units.
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