In this study we developed a method to profile phosphoproteins in human cell extracts without requiring phosphopeptide enrichment, by using a negative ionization technique of precursor scanning mass spectrometry to selectively identify phosphopeptides in negative ion mode and isolate them for sequencing. Measurement of −79 Da ion signal intensity allowed us to quantify relative changes in phosphorylation site occupancy, circumventing the need for stable isotope labeling. Thus, the method is applicable to samples which are difficult to examine by isotopic metabolic labeling, such as human tissues and fluids. We then used the method to interrogate targets of B-Raf/MKK/ERK signaling, which is constitutively active in melanoma due to oncogenic genomic mutations in B-Raf. Ninety sites were significantly altered by acute treatment of cells with MKK1/2 inhibitor. Responsive phosphoproteins included ERK and RSK enzymes as well as many signaling effectors and cytoskeletal regulators. Biochemical validation established a function for MINERVA/FAM129B, a member of a novel gene family, in controlling cell invasion into 3-dimensional extracellular matrix in a phosphorylation-dependent manner. Our findings reveal new insight into how oncogenic B-Raf controls properties of cancer cell invasion with potential relevance to metastatic behavior.
The ability to identify phosphopeptides without affinity enrichment represents a significant advance in phosphoproteomics. Peptide selection by LC-MS/MS relies on random sampling weighted by abundance, therefore phosphopeptides in complex, unenriched samples compete poorly against unphosphorylated peptides. This is because phosphopeptide abundances are often low due to substoichiometric phosphorylation, and because phosphopeptide signals are generally weak in positive mode MS, due to their acidity. Use of negative mode MS and detection of the −79 Da signature allows more selective detection and sequencing of phosphopeptide ions against the high background of unphosphorylated peptides. An advantage is that it may at least partly circumvent recovery biases seen with affinity enrichment. The Phosphosite database currently contains more than 25,000 phosphorylation sites in human proteins, most identified after enrichment using Fe3+-IMAC, TiO2, or anti-phosphotyrosine antibodies. As in most large scale studies, the phosphoproteins we observed mainly reflected high abundance proteins where multiple peptides were often detectable. This suggests that some of the 219 localized sites we identified that were not reported previously might be attributable to phosphopeptides that are normally excluded by enrichment methods.
Although the method of precursor ion scanning has been used for many years to identify phosphosites, up to now it has mainly been applied to samples of low complexity, due to limitations in instrument capabilities. A breakthrough was the introduction of the 4000 QTrap mass spectrometer, which can scan for −79 Da fragment ions and then rapidly switch polarity from negative to positive mode for MS/MS. Several advancements allowed us to extend the method to complex samples (Suppl. Methods). A critical improvement was to narrow the isolation window for positive mode scanning. Often in complex samples, the −79 Da phosphopeptide signal is simple but is followed by a more dense pattern of peaks after switching to positive mode. This is due to signals from unphosphorylated peptides alongside phosphopeptides. “Distraction” then occurs, due to preferential MS/MS of coeluting unphosphorylated peptide ions whose intensity or resolution exceeds that of the phosphopeptide ion. Therefore, it was necessary to restrict the mass window to less than 5 Da in the positive scan cycle, in order to partially compensate for this effect. In addition, when switching to positive mode, the instrument selects a positively charged m/z with the same charge state but opposite polarity to the negatively charged precursor ion. However, because the efficiency of charging is not always predictable, the positive ion selected for MS/MS may not represent the charge state with highest intensity. In our analysis, ~5,000 −79 Da precursor ion signals had intensity high enough to match and group peaks between adjacent fractions, and another ~20,000 singletons were observable. Thus, the phosphopeptides we report in this study represent only a small fraction of what we believe can be identified by precursor ion scanning. Software improvements for instrument control are needed to enable sequencing of multiple peaks within the mass window and to more accurately predict positive charge state. With improved software, precursor ion methods should yield greater data capture and representation of the phosphoproteome of complex mixtures.
Of the 90 phosphorylation sites identified in this screen by their sensitivity to U0126, 60 were maintained at an elevated level by constitutive B-Raf signaling. Sensitivity of these phosphosites to acute inhibitor treatment suggests that they are recognized by cellular phosphatases with sufficient basal activity to reverse phosphorylation within the 4 h window. These may be useful intracellular markers of MAP kinase pathway activity state. We compared our findings to a global profiling analysis of EGF-stimulated HeLa cells (, Olsen et al, 2006
) which represents the most comprehensive summary of regulated phosphorylation to date. It includes many targets of the MAP kinase pathway, a central mechanism in EGF receptor signaling. Thus, it was not surprising that 32 of the 60 sites we observed to be positively regulated in melanoma were also observed in the HeLa study, of which 16 were also upregulated by EGF stimulation. However, 16 of the phosphosites responsive in melanoma were either unresponsive to EGF or responded in the opposite direction in HeLa cells, suggesting that cells and treatments vary significantly with respect to downstream effectors and susceptibility to dephosphorylation.
The remaining 30 sites were elevated in response to MKK1/2 inhibitor. Thus, one-third of regulated phosphorylation events involved repression of phosphorylation by oncogenic B-Raf signaling, possibly through inhibition of protein kinases or derepression of protein phosphatases. This frequency was higher than that in HeLa cells, where only one-fifth of phosphorylation events decreased in response to EGF treatment (Olsen et al., 2006
), and raises the possibility that phosphorylation repression mechanisms may be sustained by B-Raf signaling in cancer in ways that differ from acute growth factor stimulation. For example, cells might induce expression of phosphatases under conditions of sustained signaling, to levels not seen after acute stimulation. Consistent with this we observe that less than 10% of ERK1/2 is activated in melanoma cells and not further enhanced by added mitogen. In contrast, more than 50% ERK1/2 is activated in NIH3T3 cells after acute mitogen stimulation (not shown). This suggests that feedback inhibitory mechanisms exist in melanoma cells, and we speculate that MKK1/2 inhibitor may enhance phosphorylation of many proteins by derepressing these mechanisms.
Although phosphotyrosine comprised 4% of phosphosites in the total dataset, except for ERK1/2 and GSK3, the majority of phosphosites responsive to inhibitor were on Ser/Thr residues. This was expected, based on the phospho-Ser/Thr selectivity of MAP kinase pathway enzymes. Unexpectedly however, acidophilic sites were also observed as regulated targets, suggesting crossregulation of casein kinase enzymes by oncogenic B-Raf/MKK/ERK. In NIH3T3 cells, casein kinase II constitutively associates with the scaffold protein, Kinase Suppressor of Ras (KSR) and contributes to activation of B-Raf and Raf-1 (Ritt et al., 2007
). Our findings raise the possibility that casein kinase II may be involved in crossregulatory signaling to additional targets in the MAP kinase pathway besides Raf.
Known ERK1/2 pathway targets identified in this study included RSK1/2, cortactin, stathmin, STAT3, hnRNPK, and MARCKS. The majority were signaling effectors and/or cytoskeletal regulators, which in part reflects the well-known bias of proteomics towards proteins of high abundance. The example of RSK1/2 illustrates the advantage of protein separation before proteolysis in order to reduce sample complexity. Here, phosphorylation of RSK isoforms could be distinguished by combining protein spectral counts with phosphopeptide peak intensity measurements, from which preferential activation of RSK2 could be demonstrated. Differential phosphorylation of protein isoforms is difficult to observe using phosphopeptide enrichment strategies, because phosphosites shared between isoforms often occur within identical peptide sequences that cannot be distinguished by mass.
Among the ~10,000 protein families in the human genome, >40% are of unknown function and not linked to known biological processes. Thus, demonstrating the biological function of unannotated genes provides an excellent way to validate the results of screening. For this study, we chose MINERVA, which belongs to a gene family of unknown category and unknown basic function, and is found widely expressed across tissues. Seven splice variants are predicted, two of which contain the C-terminal phosphorylation site cluster that we observed. The low invasion phenotype of MINERVA knockdown cells, combined with rescue by WT but not Ser→Ala mutant protein, provides convincing evidence that the protein controls events that promote movement through extracellular matrix, and that hyperphosphorylation in response to oncogenic B-Raf is necessary for this function. We speculate that inhibiting B-Raf/MKK/ERK in melanoma cells represses invasion by recruiting unphosphorylated MINERVA to cell-cell membrane junctions, and that phosphorylated MINERVA not only derepresses invasion by relocalizing away from membranes, but also regulates events which actively promote invasion. Our studies demonstrate the role of a previously unannotated gene family in cancer cell invasion.