To identify CrkL-SH3 binding proteins with potential relevance to Reelin/Dab1-signaling, we examined E16.5 murine brain extracts for CrkL-SH3 binding partners. Our approach was based on affinity chromatography using a fusion protein of GST and the amino-terminal SH3 domain of CrkL. We developed a standard pulldown assay and tested if our conditions were achieving success by performing immunoblots for known CrkL-SH3 binding partners using pulldowns of either GST-CrkL-SH3 or GST alone. We immunoblotted for five known CrkL-SH3 binding proteins, the Ras GEF SOS1, the Rap GEFs C3G and DOCK4, the Arf GAP Ddef2/ASAP2 and the adaptor protein Cin85. Each of these proteins was found bound to the GST-CrkL-SH3 resin but not to the GST resin (). To achieve levels of bound protein sufficient for mass spectrometry analysis, we scaled-up our experiment and used the strategy outlined in . Briefly, E16.5 murine brain extracts were pre-cleared with glutathione agarose and then with agarose bound to GST. The pre-cleared extract was then divided in half. One half was subjected to a pulldown using GST while the other was subjected to a pulldown using GST-CrkL-SH3. After washing, bound proteins were eluted using SDS protein sample buffer and eluates were subjected to SDS-PAGE. The eluted proteins were visualized by staining the gel with coomassie blue which indicated multiple proteins were bound to the GST-CrkL-SH3 resin which were absent from the GST resin (). Each gel lane was sectioned into 24 regions above the position of the GST-CrkL-SH3 fusion. Care was taken so as not to use the section of the gel where the GST and GST-CrkL-SH3 lanes meet. Each region was diced into 1 mm cubes, digested in-gel with trypsin, and extracted peptides were subjected to liquid chromatography tandem mass spectrometry in a linear ion trap mass spectrometer. Mass spectra were analyzed using SEQUEST30
and a concatenated forward and reverse mouse NCI protein database approach29
that facilitated filtering of top SEQUEST peptide matches to a less than 0.01% false discovery rate as described in the Experimental Procedures section for proteins identified by three or more peptides and which were not identified in GST alone analyses. These requirements eliminated all peptides whose top SEQUEST matches were to the decoy database. This resulted in the identification of 101unique proteins (Supporting Information Table 1
). All identified peptides for each of these proteins are listed in Supporting Information Table 2
. As Crk/CrkL function within the cytosol we conducted a bioinformatic analysis to determine the cellular compartment of the identified CrkL-SH3 binding proteins using MGI35
as way to determine if certain substrates were more or less likely to interact with CrkL in vivo
. When compartmental information was not available in the MGI resource, we used the program LOCATE36
, and if neither of these programs contained information for a given protein we examined individual articles. We found no compartmental information for only four proteins (see Supporting Information Table 2
). These analyses revealed six proteins that were not reported to have at least some contact with the cytosol: (1) SAM and SH3 domain containing 1, serine/arginine repetitive matrix 1; (2) euchromatic histone methyltransferase 1; (3) X-ray repair complementing defective repair in Chinese hamster cells 1; (4) NCK interacting protein with SH3 domain; (5) PHD finger protein 8, protein phosphatase 1; (6) regulatory subunit 10, splicing factor 3b, subunit 2. These six proteins were deemed exclusively nuclear. However, it is worth noting that some details regarding cellular compartments may not be complete, as the protein NCK interacting protein with SH3 domain is so named given its ability to interact with NCK, a cytosolic protein that is part of the greater Crk/CrkL adapter protein family. Thus >91% of the CkrL-SH3 binding proteins identified in our study are not excluded from the cytosol and therefore and interaction with Crk/CrkL.
Purification of CrkL-SH3 binding partners from embryonic murine brain extract
A Venn diagram is shown in comparing the overlap of our list of CrkL-SH3 binding proteins from embryonic brain to the 21 previously known Crk/CrkL-SH3 binding proteins whose interactions were shown biochemically using extracts from various cells or tissues (Supporting Information Table 3
). We identified in our analysis 11/21 known Crk/CrkL-SH3 binding partners. The reasons for not identifying eight of the other ten can largely be explained: three of the binding partners are predominantly or exclusively expressed in hematopoietic cells (Map4k1, Dock2 and Cd34), two were identified but eliminated from our list as they were also identified in our GST alone control (Eef1a2 and Map4k5), one (Rac1) was not identified due to its size being less than the GST-CrkL-SH3 domain, below which we did not analyze, one (NS1) was from the Spanish Influenza A virus, and one (Kalrn) was likely too large (340kDa) to be identified in our gel-based analysis. The reasons for not identifying the other two (Kidins220 and Mapk8) cannot be fully explained. Also included in the Venn diagram are 64 proteins that were singled out as potential Crk-SH3 interacting proteins following a binding reaction of His6
-Crk-SH3 to an array of 1,536 putative SH3 binding peptides37 (Supporting Information Table 4
). Eleven (52%) of the known Crk/CrkL-SH3 binding proteins, and ten (16%) of the peptide array-based Crk-SH3-binding proteins overlapped with our dataset. However, eighty six (86%) of the proteins in our dataset have not previously been reported to interact with Crk/CrkL. This could be explained as our study is the first large-scale analysis of CrkL-SH3 binding proteins from tissue, the fact that we are using a unique embryonic tissue type, as well as the increased sensitivity of today’s mass spectrometers. Supporting Information Table 6
provides a list of the overlapping proteins in the Venn diagram.
Bioinformatic Characterizaton of Crk/CrkL-SH3 binding proteins
Given that GST binding proteins were eliminated from the dataset, the CrkL-SH3 binding proteins reported here are arguably of only two types: proteins showing novel and direct binding to the CrkL-SH3 domain or proteins that bind indirectly to the CrkL-SH3 domain. An indication that the proteins in our dataset contain a number of proteins directly binding to the CrkL-SH3 domain would be if they are enriched in the target domain known to bind to Crk-SH3. We assessed this in two ways. We first conducted a motif-x analysis31
. Motif-x facilitates the identification of motifs enriched within a defined set of proteins relative to the abundance of the same motif in the proteome of the organism under investigation. Among other proline-containing motifs, we found in our CrkL-SH3 binding protein dataset a greater than 12-fold enrichment of a motif similar to the optimal Crk-SH3 binding motif extracted from the motif scanning tool at Scansite23
(). Second, we subjected each individual protein sequence in the three Crk/L-SH3 datasets from to a Scansite23
motif analysis to determine the number and percent of proteins containing a potential Crk/CrkL-SH3 binding motif. summarizes the results and each dataset shows a similar profile in number and percent of each stringency type of Scansite-predicted Crk-SH3 binding motifs. Indeed the three datasets show 92%, 94% and 95% of proteins from the dataset presented here, the dataset from the PATS analysis and the dataset from the known Crk/CrkL binding proteins have at least one predicted Crk-SH3 binding domain respectively. 20/21 of the known proteins contained a Scansite-identifiable Crk-SH3 binding motif. The one protein, Rac1, in which a Crk-SH3 binding domain was not identified by Scansite was shown to bind to the Crk-SH3 domain by virtue of a PPP
Rk motif in the C-terminal region of Rac138
, with the PPP and RKR sequences shown to be essential, at least as one set or another by mutating each set to a series of alanines. Thus, in this case, it appears that the first proline and the last lysine (underline above) in the PXXPXK motif proved sufficient for binding to the Crk-SH3 domain. Given that the proteins identified in this study have enrichments in Crk/CrkL-SH3 binding motifs and given 92% of the identified proteins have at least one Crk/CrkL-SH3 binding motif as predicted by Scansite, this suggests the embryonic brain CrkL-SH3 binding proteins are enriched in proteins that bind directly to the CrkL-SH3 domain.
Summary of Proline-Based Motifs Identified from Crk/CrkL-SH3 binding proteins data sets
To determine if the CrkL-SH3 binding proteins we identified were enriched for biological processes consistent with potential functions of Reelin signaling we used the gene ontology analysis program Panther33
. For the purpose of generating a basis of comparison we also performed the Panther analysis on the top 176 proteins identified in a large-scale proteomic analysis of a whole cell extract of E16.5 murine brain34
. The Panther program calculates the percentage of the annotated proteins in the dataset falling into one of 16 biological process categories with some proteins conceivably being part of more than one category. The complete Panther analysis we performed for each dataset is presented in Supporting Information Table 5
. In we graphically portray the 16 categories and show the percent of proteins from each of the datasets falling into each category. When we compared the CrkL-SH3 proteins identified in this study to the top 180 most identified proteins from the embryonic brain extract to we found that the CrkL-SH3 binding proteins had two times the representation in the cell communication and cell adhesion PANTHER categories. Conversely, the CrkL-SH3 binding proteins were more than two-fold less enriched in the category “Generation of precursor metabolites and energy.” A similar reduction was also observed in “Metabolic processes.” Supporting Information Table 5
lists the CrkL-SH3 binding proteins in each category. Intriguingly, an enrichment of CrkL-SH3 binding proteins in cellular communication and the regulation of cell adhesion is consistent with cellular mechanisms at play when cells are responding to signaling cues regulating cellular migration or positioning. Given that one of these proteins, C3G, was previously shown to be phosphorylated on tyrosine in a Reelin and Crk/CrkL-dependent manner, likely due to activated SFKs, we asked if other proteins in these categories might have predicted Src target motifs using Scansite23
. Intriguingly, 80% of the proteins in these categories had putative Src phosphorylation sites as opposed to 69% of the entire dataset.
It is important to note that proteins in categories where enrichments were not observed may still be important to Reelin signaling. For example, proteins associated with the Golgi may be classified in the transport category, and while this may not seem immediately relevant to Reelin signaling, Reelin was recently reported to dramatically alter Golgi polarity39
. However, while regulated intracellular trafficking may be initiated by Crk/CrkL-SH3 binding proteins, downstream trafficking effector proteins may not necessarily be found bound directly to the Crk/CrkL-SH3 domain. An example of Reelin regulated vesicular trafficking was recently reported describing Reelin-induced Cdc42 activation of N-WASP and the Arp2/3 complex40
. Of note, not only was N-WASP previously identified as a pY-Dab1 binding protein41
, we also identified N-WASP to be a novel CrkL-SH3 binding protein.
Given that we identified a number of novel CrkL-SH3 binding proteins we conducted again a small-scale GST-CrkL-SH3 pulldown followed by immunoblotting for five of the novel proteins that may have particular relevance to Reelin signaling, ARAP1, LPD, TKS4, LPP and N-WASP. The results are shown in and provide confirmation that these proteins bind to GST-CrkL-SH3 and not to GST alone. ARAP1 is a protein with Arf GAP, Rho GAP, Ankyrin repeat, Ras-associating (RA), and Plekstrin homology (PH) domains and is thought to function at the interface of various signaling activities. Indeed ARAP1 is known to have PI3,4,5
-dependent Arf-Gap activity and plays roles in regulating receptor recycling and Golgi structure42, 43
. Given Reelin is also thought to be involved in regulating actin and golgi dynamics and Reelin-induced PI3K activity appears critical for multiple aspects of Reelin signaling28, 44, 45
ARAP1 is an intriguing, albeit complex candidate in Reelin signaling. LPD (Lamellipodin) is an Ena/VASP binding protein that regulates actin dynamics and induces the formation of lamellipodia requiring the PI3K product PI3,4
and is therefore another intriguing potential effector of Reelin signaling46, 47
. TKS4 (Tyrosine Kinase Substrate with 4 SH3 domains) is important in the formation of actin rich podosome structures that recruit matrix metalloproteases48, 49
. Best understood in cancer paradigms, podosomes are also being studied in the context of non-cancerous migratory cell types50, 51
. Intriguingly TKS4 requires phosphorylation by SFKs for its activity and binds the PI3K lipid products PI3
P and PI3,4
via its Phox homology (PX) domain49, 52
. LPP (lipoma preferred (translocation) partner) is a member of the zyxin family of Ena/VASP binding proteins. LPP localizes to sites of cell adhesion and is thought to recruit Ena/VASP proteins for localized actin polymerization and cell protrusion53, 54
Biochemical Characterization of CrkL-SH3 binding proteins
In order to determine if the CrkL-SH3 binding partners we identified might also interact with other SH3 domains, we directly compared the binding of three of the known and three of the novel CrkL-SH3 binding proteins in pulldown assays using the CrkL-SH3 (N-terminal) domain and SH3 domains from five other proteins. Strikingly, only one substrate showed a dominant preference for only one SH3 domain, that being C3G for the CrkL-SH3 domain (). While highly specific binding preferences can greatly facilitate our understanding of specificity in signaling, we argue that even if some substrates do not bind exclusively to the CrkL-SH3 domain this does not exclude them from contributing to Reelin or other CrkL-dependent signaling pathways. However, our data suggest that one should not assume that the CrkL-SH3 binding proteins that we have identified bind exclusively to CrkL’s N-terminal SH3 domain.
One intriguing possibility is that Reelin-Dab1 signaling can differ at different stages of development or in different tissues depending on the abundance and availability of the various CrkL-SH3 binding proteins. We tested age-specific binding differences for five proteins to the CrkL-SH3 domain from extracts of E16.5, P0 and P21 murine brains. While the majority of differences in binding appear to be due to differences in the individual protein levels in the various brain extracts, this was not true for LPP, TKS4 and N-WASP (). While LPP increased in protein expression from E16.5 to P21 its binding to the CrkL-SH3 domain at P21 was much reduced compared to the binding at E16.5. TKS4 also showed reduced binding from P21 extracts even though the levels of TKS4 didn’t change. Conversely, while N-WASP levels changed little across the three stages, its binding to the CrkL-SH3 domain increased at older stages. These data may be explained by a regulated interaction of some CrkL-SH3 binding partners. Regulated binding to SH3 domains has precedence, with an excellent example being ERK1/2-dependent phosphorylation of SOS1 leading to a disruption of the binding of SOS1 to the SH3 domain of Grb255
. Alternatively, the observed differences could be due to CrkL-SH3 binding partners in some brain extracts being stably associated with either endogenous Crk/L or other proteins such that they are inaccessible during the pulldowns. In addition, should the level of total CrkL-SH3 binding partners at one stage of development exceed the number of GST-CrkL-SH3 in the pulldown this could also give the perception of stage-specific differential binding. Given ~20 μg of GST-CrkL-SH3 fusion protein and 4 mg of extract was used in the small-scale pulldowns the GST-CrkL-SH3 fusion protein would be in excess until the sum of the CrkL-SH3 binding partners in the extract achieved 0.5% of the total protein. Therefore this issue is not likely to be an important factor in our analyses.
While significant work remains to fully characterize each of the identified CrkL-SH3 binding proteins, particularly their possible roles in Reelin signaling, we have further characterized one of the interacting proteins, LPP, by examining complex formation using co-immunopreciptiation. HEK 293 cells were transfected with either a Dab1 wildtype construct or a mutant Dab1 construct (Y5F). The Y5F construct has five tyrosine-to-phenylalanine mutations such that Dab1 cannot be tyrosine phosphorylated. Additionally, each of the Dab1 constructs is fused to an FKBP dimerization domain that can be used to induce dimerization with the bivalent compound AP20187. Importantly, this leads to tyrosine phosphorylation in the case of the wildtype Dab1 construct and not the mutant15
. After treating both sets of transfected cells with AP20187, LPP was immunoprecipitated and a trimeric complex of LPP, CrkL and phosphotyrosyl-Dab1 was observed, whereas in the case of the Y5F construct only the LPP-CrkL dimeric complex was observed, showing a trimeric complex dependent on Dab1 tyrosine phosphorylation ().