The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.

INTRODUCTION

Lyn is a member of the Src family of intracellular membrane-associated tyrosine kinases [SFKs (Src-family kinases)] that control many cellular processes, including the transformed phenotype of several cancers and leukaemias. SFKs are involved in several signal transduction pathways from cytokine/growth factor receptors, immune receptors and integrins [1]. SFKs can also directly activate STATs (signal transducer and activator of transcriptions), particularly in transformed cells [2]. Several studies have illustrated the important role of Lyn in both CML (chronic myeloid leukaemia) and AML (acute myeloid leukaemia) cells [37]. Primary AML cells have elevated Lyn kinase activity [3], and, although the BCR (breakpoint cluster region)–Abl fusion protein is the initiating molecule for CML, there is a crucial down-stream role for Lyn in BCR–Abl-induced leukaemogenesis [5,6]. Previously, it has been shown that in B-NHL (B-non-Hodgkin's lymphomas) there is a signalling complex containing Lyn and STAT3 that is formed through the scaffolding protein Cbp [Csk (C-terminal Src kinase)-binding protein], and this appears essential for their survival [8].

Several investigations have implicated a significant role for Lyn in the development of certain solid tumours. Lyn has been detected in colorectal tumours that are metastatic, but not at earlier stages of cancer development [9]. Lyn also regulates signalling mechanisms in prostate cancer cells, including LNCaP and DU145 cells, where it is involved in cell migration [10], and breast cancer cell lines, including MCF-7 cells where overexpression of Ctk/CHK (checkpoint kinase) can lead to decreased Src and Lyn activity and expression, resulting in reduced growth, transformation and invasion [11]. Lyn is expressed in normal prostate epithelium and Lyn−/− mice have significantly impaired development of this gland with reduced size and ductal networks [12]. When primary prostate cancer tissues were examined, 95% showed Lyn expression and almost always at higher levels than in normal tissue. Significantly, inhibition of Lyn in prostate cell lines, including DU145, resulted in reduced proliferation, not only in vitro, but also in mice carrying prostatic cancer xenografts [12]. Recent proteomic analysis of 130 tumour cell lines illustrated that Lyn was one of the most common activated tyrosine kinases present and points to the potential utility of anti-Lyn/SFK therapy for the treatment of numerous neoplasms [13].

Two main mechanisms for post-translational control of Src kinases exist [14]: (i) altering the phosphorylation status of the kinase and (ii) polyubiquitination, followed by degradation. The tyrosine kinase Csk promotes inactivation of Src kinases by phosphorylating the negative regulatory C-terminal tyrosine residue [15]. Cbp [16], an SFK-binding scaffold protein [17], is a key molecule in reducing SFK/Lyn kinase activity, by recruiting the inhibitory kinase Csk via phosphorylation of Tyr314 within Cbp, which binds to the SH2 (Src homology 2) domain of Csk. Our results also demonstrated that Cbp can act as a crucial adaptor that co-ordinates both kinase inactivation and ubiquitination/degradation of Lyn, through the phosphorylated Tyr314 motif of Cbp also binding to the SH2 domain of the E3 ubiquitin ligase SOCS1 (suppressor of cytokine signalling 1) [17]. Members of the SOCS family bind elongins B and C via their SBs (‘SOCS boxes’), which in turn mediate polyubiquitination of SOCS-associated proteins prior to their degradation within proteasomes [18]. Our previous data have shown that when Lyn is over-active, as can been found in several cancers/leukaemia, it induces STAT5 activation leading to SOCS1 induction, and in the presence of Cbp leads to enhanced polyubiquitination/degradation of Lyn [19]. Cbp also interacts with other signalling molecules, potentially co-ordinating multiple SFK regulated pathways through pY (phospho-tyrosine)-mediated SH2 interactions [19,20].

The utility of SOCS molecules to down-regulate signalling as potential therapeutic agents was shown by Jo et al. [21], who utilized a recombinant cell-penetrating SOCS3 protein to attenuate pro-inflammatory signalling in mice. Using our knowledge of the Lyn/Cbp/SOCS1 pathway in regulating Lyn/SFK we sought to generate fusion proteins of the minimal functional motifs and domains of Cbp and SOCS1 to make molecules with enhanced Lyn/SFK-degrading capacity that would be dependent upon the activity of Lyn/SFK for their ability to degrade Lyn/SFK, independent of endogenous Cbp and SOCS1. In the present study we show that isolated tyrosine motifs from Cbp are phosphorylated by active Lyn, mediate binding to Lyn with high affinity and when fused to the SB of SOCS1 enhance the polyubiquitination/degradation of Lyn. By expressing this fusion Cbp/SOCS1 protein in the DU145 cancer cell line known to require Lyn for important signalling pathways, we find reduced Lyn expression, reduced caspase 3, appearance of an apoptotic phenotype and inhibition of their growth/expansion.

MATERIALS AND METHODS

Plasmid construction

All plasmid constructs were generated by site-directed mutagenesis using oligonucleotides (sequences available upon request) and subcloned into the appropriate vector and confirmation by sequencing. The Myc-tagged Cbp expression constructs (pCMV-Cbp [16]) were generously provided by Dr M. Okado (Osaka University, Japan). The construction of the murine Lyn expression plasmids pCA-Lynwt, pCA-LynY397F, pCA-LynY508F [22] and murine Lyn SH2 domain VP16 fusion plasmid pVP16-SH2 [23,24] have been described in detail previously [25]. The rat Cbp LexA fusion constructs pBTM116-Cbp, pBTM116-CbpΔ352, pBTM116-CbpPhe381/Phe409, pBTM116-CbpΔ352Phe381, pBTM116-CbpΔ352Phe409, pBTM116-CbpΔ352Phe381/Phe409 were generated using pCMV-Cbp as a template and subcloning into pBM116 [26] in-frame with LexA as described previously [17], for use in the pY Y2H (yeast two-hybrid) system [17]. Generation of the HA (haemagglutinin)-tagged Ub mammalian expression vector has been described previously [17]. The SH2 domains of Src, Hck, Lck, Fyn, Yes, Fgr, Frk, Brk, Blk and SrmS were cloned from cDNA isolated from fetal liver (E13) erythroid progenitors of mice by PCR using oligonucleotides designed to each SH2 domain (sequences available on request) and directionally subcloned into pVP16, generating VP16 fusions for use in the pY Y2H system [17]. The SH2 domains of Lyn, Hck and Src were also subcloned into the His-NusA-His-tag bacterial expression vector pET-44TEVa [derived from pET-44a (Novagen) with replacement of the thrombin/enterokinase cleavage sites with a TEV (tobacco etch virus) cleavage site, details available on request].

The constructs containing an eGFP [enhanced GFP (green fluorescent protein)] fusion with different phosphorylatable tyrosine motifs of Cbp for mammalian expression, or their non-phosphorylatable phenylalanine mutants, were generated by subcloning annealed oligonucleotide pairs into pEGFP-C1 or pEGFP-C2 (Clontech), generating pEGFP-Tyr314, pEGFP-Phe314, pEGFP-Tyr314/Tyr409 and pEGFP-Phe314/Phe409 (see Figures 1A and 1B for details of encoded amino acids). The constructs producing eGFP fusions of the C-terminal region of Cbp (amino acids 352–424), encompassing Tyr381 and Tyr409, or these two tyrosine residues mutated to phenylalanine, were generated by subcloning this region from pCMV-Cbp and pCMV-CbpPhe381/Phe409 [17] respectively, into pEGFP-C2. Addition of the SB of SOCS1 (amino acids 170–211) to the constructs pEGFP-Cbp352-C and pEGFP-Cbp352-CPhe381/Phe409 was achieved by subcloning this region from pEF-SOCS1 [provided by Dr S. Nicholson, Walter and Eliza Hall Institute (WEHI), Melbourne, Australia]. The SB was also subcloned into pEGFP-C2 on its own.

Schematic diagrams of Cbp and SOCS1, and fusion construct details

Figure 1
Schematic diagrams of Cbp and SOCS1, and fusion construct details

(A) Schematic of motif/domain architecture of Cbp and SOCS1. Amino acids are numbered; the transmembrane (TM) region, proline-rich region (PP), palmitoylation motif (CC) and pYs of Cbp are indicated. Binding sites for Csk, SOCS1, Lyn and SFKs are shown. The SH2 and SB of SOCS1 are indicated, which bind Cbp/JAK (Janus kinase) and elongin B/C respectively. (B) Detail of the eGFP fusion constructs utilized and the regions of Cbp/SOCS1 they encompass. Constructs were generated by subcloning annealed oligonucleotide pairs corresponding to the tyrosine/phenylalanine motifs, or the indicated regions of Cbp/SOCS1 into pEGFP-C1/2 (Clontech) and were confirmed by sequencing.

Figure 1
Schematic diagrams of Cbp and SOCS1, and fusion construct details

(A) Schematic of motif/domain architecture of Cbp and SOCS1. Amino acids are numbered; the transmembrane (TM) region, proline-rich region (PP), palmitoylation motif (CC) and pYs of Cbp are indicated. Binding sites for Csk, SOCS1, Lyn and SFKs are shown. The SH2 and SB of SOCS1 are indicated, which bind Cbp/JAK (Janus kinase) and elongin B/C respectively. (B) Detail of the eGFP fusion constructs utilized and the regions of Cbp/SOCS1 they encompass. Constructs were generated by subcloning annealed oligonucleotide pairs corresponding to the tyrosine/phenylalanine motifs, or the indicated regions of Cbp/SOCS1 into pEGFP-C1/2 (Clontech) and were confirmed by sequencing.

SH2 cloning, expression and purification for peptide affinity assays

The Src-SH2 sequence (corresponding to residues 144–249 of human Src; UniProt P12931) was cloned into the pET-21d vector between the NcoI and XhoI sites. The PCR template sequence was a synthetic human c-Src gene construct (Mr Gene). The Lyn-SH2 sequence (corresponding to residues 122–227 of human Lyn; UniProt P07948) was cloned into the pET-31b vector between the NdeI and XhoI restriction sites. The PCR template sequence was human Lyn in pJP1520 (Harvard Plasmid Database clone HsCD00037974). The pET-14b Hck-SH2 construct, encoding residues 119–224 of human Hck (p59 isoform; UniProt P08631-2) was a gift from John R. Engen of Northeastern University, Boston MA, U.S.A. [27]. The integrity of the coding sequence for each SH2-expressing plasmid was confirmed by DNA sequencing. All of these constructs express the SH2 domain as the respective native sequence with an N-terminal methionine residue due to the 5′-ATG start codon. The SH2 domains were expressed in BL21 (DE3) Escherichia coli cells and were purified from the cell lysate by affinity chromatography on O-phospho-L-tyrosine agarose (Sigma), followed by size-exclusion chromatography on Superdex 10/300 (GE Healthcare), as described previously [28,29]. Protein concentrations were estimated from the absorbance at 280 nm, using an molar absorption coefficient (ϵ) calculated from respective amino acid sequence using ProtParam [30].

MS

The molecular masses of purified proteins were determined using 6510 Q-TOF–LC/MS (quadrupole–time-of-flight–liquid chromatography/MS) from Agilent Technologies. Purified SH2 samples in 10 mM phosphate, pH 7.5, and 1 mM DTT (dithiothreitol) were first loaded on a C18 guard column and then eluted into the mass spectrometer using a 10 min gradient of 28.5–95% acetonitrile, containing 0.1% formic acid in MilliQ H2O. Mass spectra were deconvoluted using the Mass Hunter software from Agilent Technologies.

CD spectroscopy

CD spectra were measured in 10 mM phosphate buffer, pH 7.5, and 1 mM DTT using an Aviv Model 410 SF CD spectrometer. Spectra were recorded from SH2 samples at 0.1–0.2 mg/ml using a 1 mm pathlength quartz cuvette at 20°C. CD data were collected from 250 to 190 nm at 100 nm/min in continuous scanning mode and with a data-pitch of 0.5 nm. Final CD spectra were obtained by averaging three scans and subtracting the buffer spectrum. The reversibility of thermal denaturation was assessed by measuring the CD spectrum at 20°C, after heating to 90°C and then again after cooling to 20°C. Thermal denaturation studies were performed by monitoring the ellipticity at a fixed wavelength (Lyn-SH2, 227 nm; Hck-SH2, 228 nm; and Src-SH2, 218 nm) while heating the sample from 25 to 90°C at 1°C/min. The non-linear regression was used to determine the melting temperature (Tm) from ellipticity compared with temperatures curves, as described previously [31].

Peptide affinity measurements

Phospho-tyrosyl peptides were purchased from GLBiochem. All peptides were synthesized with a FITC group attached to their N-terminus via an aminocaproic acid linker and had an amidated C-terminus. FITC–HMTA corresponded to residues 321–331 of the hamster polyomavirus middle T-antigen (UniProt P03079) with a pY at position 324 (FITC–Acp-EPQpYEEIPIYL). FITC–Cbp381 corresponded to residues 384–394 of human Cbp (UniProt Q9NWQ8) with pY at position 387 (FITC-Acp-EPDpYEAIQTLN). FITC–Cbp409 corresponded to residues 414–424 of human Cbp with pY at position 417 (FITC–Acp-ENDpYESISDLQ). Peptides were dissolved in DMSO at 1 mg/ml and then diluted into protein samples in 50 mM phosphate, pH 7.5, 100 mM NaCl and 1 mM DTT to a final peptide concentration of approximately 20 nM (~1/25000 dilution). Fluorescence anisotropy measurements were made at 20°C using a Cary Eclipse fluorescence spectrophotometer with automated polarizers. Dilution titrations were performed and equilibrium dissociation constant (Kd) values were calculated by non-linear regression of FITC anisotropy compared with protein concentration data, as described previously [32].

Cell culture and transfection

COS-7, LNCaP, DU145 and MCF-7 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) with 10% FCS (fetal calf serum). COS-7, LNCaP, MCF-7 and DU145 cells were transiently transfected with plasmids using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Transfection efficiencies were routinely >80% for single plasmids and >60% for transfections with two plasmids for COS-7 cells, whereas DU145 showed ~30% transfection efficiency, with LNCaP and MCF-7 cells showing ~10–20% efficiencies. Individual plasmids were titrated in transfections to achieve equivalent expression levels as determined by immunoblotting. LNCaP, DU145 and MCF-7 cells were transfected with linearized plasmids using Lipofectamine™ 2000 as described above for COS-7 cells. Transfected LNCaP, DU145 and MCF-7 cells were selected in G418 (Invitrogen) (1 mg/ml) 48 h after transfection for 7–10 days to isolate stable transfected cells. Kill-curves for these three cell lines using G418 showed a concentration of 1 mg/ml produced ~100% cell death of un-transfected cells after 7 days.

pY-specific Y2H assays

The Y2H system [23,26] utilized the Saccharomyces cerevisiae L40 and Y187 strains. The pY-specific version we employed has previously been described where in essence the L40 or Y187 strain is co-transfected with the plasmid pRSADE-MET-Lyn expressing the kinase domain of Lyn, through a methionine-regulated promoter with selection via the Ade2 gene [17]. Liquid LacZ assays were performed with, and without, the addition of methionine (200 mg/l), which suppresses the MET25 promoter driving the expression of the Lyn kinase to delineate pY-specific interactions as described previously [17].

Cell lysis, immunoprecipitation and immunoblotting

Cells were lysed in raft lysis buffer [150 mM NaCl, 1% IGEPAL CA-630 (Sigma–Aldrich), 0.5% n-dodecyl-β-D-maltoside (Sigma–Aldrich), 0.2% octyl-D-glucoside (Sigma–Aldrich), 20 mM Tris/HCl, pH 8.0, 0.1 mM Pefabloc, 10 mg/ml aprotinin, 2 mM benzamidine, 2 mM vanadate, 1 mM EDTA, 1 mM EGTA and 10 mM β-glycerol phosphate], and immunoprecipitations and immunoblotting were performed essentially as described previously [22,24] using Protein G beads (Sigma–Aldrich). Antibodies used were anti-Lyn (sc15), anti-pY (sc7020), anti-GFP (sc-8334) (Santa Cruz Biotechnology), anti-pY 4G10-HRP (horseradish peroxidase), anti-myc 4A6 (Millipore) and anti-β-actin (Abcam). Secondary antibodies were coupled to HRP (GE Healthcare) and detected by enhanced chemiluminescence (Millipore).

Fluorescent microscopy

Transfected and selected (≥7 days with G418) LNCaP, DU145 and MCF-7 cells were examined for eGFP expression on an Olympus IX71 microscope using an Olympus DP70 camera (×20) for fluorescence (eGFP filter) and by phase contrast [DIC (differential interference contrast)].

RESULTS

Rationale for fusion protein design

The scaffold protein Cbp (Figure 1A) possesses a small extracellular domain (17 amino acids), a single transmembrane domain immediately followed by two palmitoylation sites. The cytoplasmic domain contains a proline-rich motif that interacts with the SH3 domain of Lyn and a C-terminal PDZ domain-binding motif. Furthermore, there are nine pY motifs, several of which have been shown to bind specific SH2 domains, including ras-GAP (GTPase-activating protein), PI3K (phosphoinositide 3-kinase) p85R, PLCγ (phospholipase Cγ), Csk, SOCS1 and SFKs (including Lyn) [14,19]. We utilized the Lyn/SFK-binding pTyr381 and pTyr409 motifs of Cbp in our fusion constructs, as we had previously shown they are the preferred substrates of active Lyn and have a high apparent specificity/affinity for the SH2 domain of Lyn [17,19].

The SOCS1 molecule (Figure 1A) contains an SH2 domain and an SB, the latter binds the elongin B/C complex, enabling it to mediate its E3 ubiquitin ligase activity and promote the poly-ubiquitination of proteins associated with SOCS1, predominantly through its SH2 domain.

Using our knowledge of the Cbp-mediated regulation of Lyn/SFKs through Csk and SOCS1 [17,19], we sought to design constructs to direct the inactivation/degradation of Lyn/SFKs in transformed cells that are known to utilize Lyn/SFK signalling for essential aspects of their biology. We generated various eGFP fusions with the Lyn-binding pY motifs of Cbp (pTyr381 and pTyr409), the Csk binding pY motif (pTyr314), and/or the elongin B/C-binding SB domain of SOCS1 (see Figure 1B). Control constructs contained point mutations of the pY motifs, changing the tyrosine to the non-phosphorylatable phenylalanine.

Specificity and affinity of the pTyr381 and pTyr409 motifs of Cbp

The Tyr381 and Tyr409 motifs of Cbp have sequence homology to sites known to bind several SFK SH2 domains [19]; consequently we tested these sites for their degree of specificity towards Lyn and other SH2 domains (Figure 2). Using our pY-specific Y2H system [17], we found that indeed all of the SFK SH2 domains could bind full-length Cbp (Figure 2A). No reporter activity was observed with an unrelated SH2 domain (Nck2, Vav2) or when the kinase domain of Lyn was not expressed within the pY-Y2H system (results not shown). When we mutated just the Tyr381 and Tyr409 motifs of Cbp (leaving the seven other pY motifs of Cbp intact), it profoundly ameliorated the ability of the SH2 domains of Lyn, Src and Hck to bind Cbp. The SH2 domains of Lck and SrmS showed a strong, but less pronounced (than Lyn/Src/Hck), reduction in their ability to bind Cbp when the Tyr381/Tyr409 sites were mutated. Other SFK SH2 domains (Fyn, Yes, Fgr, Frk, Brk and Blk) displayed only minor dependence upon these motifs for binding to Cbp (Figure 2A).

Determining the specificity and affinity of the Tyr381 and Tyr409 motifs of Cbp for the SH2 domains of SFKs

Figure 2
Determining the specificity and affinity of the Tyr381 and Tyr409 motifs of Cbp for the SH2 domains of SFKs

(A) Lyn, Src and Hck show the highest specificity for the Tyr381/Tyr409 motifs within full-length Cbp. pY-specific Y2H (pY-Y2H) liquid LacZ assays [17] of the different SFK SH2 domains interacting with full-length Cbp (amino acids 1–424) containing the Tyr381 and Tyr409 motifs or with both these sites mutated (Phe381/Phe409). Reporter activity of the Phe381/Phe409 construct is expressed relative to that observed for the wild-type construct. Assays were performed in triplicate; means±S.D. are shown. Significantly reduced LacZ activity of Phe381/Phe409 constructs relative to Tyr381/Tyr409 constructs is indicated (*P< 0.01). LacZ activity of Phe381/Phe409 constructs for Lyn, Hck and Src SH2 domains are not significantly different, as indicated (Δ). (B) Lyn, Src and Hck have the highest specificity for the Tyr409 motif of Cbp in the pY-Y2H system. Assays were undertaken as in (A), using a C-terminal region of Cbp (amino acids 352–424) encompassing the Tyr381/Tyr409 motifs. The Tyr381 and Tyr409 motifs were mutated individually or together as indicated. Relative LacZ activity of Tyr381/Phe409 constructs that is significantly different to Phe381/Tyr409 constructs is indicated (*P< 0.01). (C) Lyn SH2 domain binds the pTyr409 peptide (FITC–Cbp409) with high affinity, comparable with that of the high affinity SFK SH2 binding peptide (FITC–HMTA), whereas the pTyr381 peptide (FITC–Cbp381) binds with lower affinity. Fluorescence anisotropy measurements were used to determine the affinity of the Lyn-SH2 domain for the FITC–HMTA, FITC–Cbp381 and FITC–Cbp409 phospho-tyrosyl peptides. Anisotropy data were fitted to a simple single binding site model to determine the minimum anisotropy, maximum anisotropy and Kd values. For clarity, data were transformed to fraction bound compared with protein concentration. Experimental data (FITC–HMTA, filled circles; FITC–Cbp381, open circles; and FITC–Cbp409, filled triangles) and the fitted binding curves (FITC–HMTA, solid line; FITC–Cbp381, dotted line and FITC–Cbp409, dashed line) are shown. (D) Lyn shows a higher capacity to interact with phosphorylated Cbp from cell lysates compared with Src or Hck. In vitro pull-down assays using purified NusA fusions of the SH2 domains of Lyn, Src and Hck with in vivo phosphorylated full-length Cbp from COS-7 cell lysates. NusA fusion proteins were purified on Ni-affinity columns (GE Healthcare) using a Profinia instrument (Bio-Rad Laboratories) with desalting according to the manufacturer's instructions. Pull-down assays [17] were run on SDS/PAGE and transferred on to PVDF membrane, then probed with anti-pY-HRP antibodies [17] and subsequently stained with Coomassie Blue to detect the NusA fusion. Quantification and statistical analysis of replica blots is shown.

Figure 2
Determining the specificity and affinity of the Tyr381 and Tyr409 motifs of Cbp for the SH2 domains of SFKs

(A) Lyn, Src and Hck show the highest specificity for the Tyr381/Tyr409 motifs within full-length Cbp. pY-specific Y2H (pY-Y2H) liquid LacZ assays [17] of the different SFK SH2 domains interacting with full-length Cbp (amino acids 1–424) containing the Tyr381 and Tyr409 motifs or with both these sites mutated (Phe381/Phe409). Reporter activity of the Phe381/Phe409 construct is expressed relative to that observed for the wild-type construct. Assays were performed in triplicate; means±S.D. are shown. Significantly reduced LacZ activity of Phe381/Phe409 constructs relative to Tyr381/Tyr409 constructs is indicated (*P< 0.01). LacZ activity of Phe381/Phe409 constructs for Lyn, Hck and Src SH2 domains are not significantly different, as indicated (Δ). (B) Lyn, Src and Hck have the highest specificity for the Tyr409 motif of Cbp in the pY-Y2H system. Assays were undertaken as in (A), using a C-terminal region of Cbp (amino acids 352–424) encompassing the Tyr381/Tyr409 motifs. The Tyr381 and Tyr409 motifs were mutated individually or together as indicated. Relative LacZ activity of Tyr381/Phe409 constructs that is significantly different to Phe381/Tyr409 constructs is indicated (*P< 0.01). (C) Lyn SH2 domain binds the pTyr409 peptide (FITC–Cbp409) with high affinity, comparable with that of the high affinity SFK SH2 binding peptide (FITC–HMTA), whereas the pTyr381 peptide (FITC–Cbp381) binds with lower affinity. Fluorescence anisotropy measurements were used to determine the affinity of the Lyn-SH2 domain for the FITC–HMTA, FITC–Cbp381 and FITC–Cbp409 phospho-tyrosyl peptides. Anisotropy data were fitted to a simple single binding site model to determine the minimum anisotropy, maximum anisotropy and Kd values. For clarity, data were transformed to fraction bound compared with protein concentration. Experimental data (FITC–HMTA, filled circles; FITC–Cbp381, open circles; and FITC–Cbp409, filled triangles) and the fitted binding curves (FITC–HMTA, solid line; FITC–Cbp381, dotted line and FITC–Cbp409, dashed line) are shown. (D) Lyn shows a higher capacity to interact with phosphorylated Cbp from cell lysates compared with Src or Hck. In vitro pull-down assays using purified NusA fusions of the SH2 domains of Lyn, Src and Hck with in vivo phosphorylated full-length Cbp from COS-7 cell lysates. NusA fusion proteins were purified on Ni-affinity columns (GE Healthcare) using a Profinia instrument (Bio-Rad Laboratories) with desalting according to the manufacturer's instructions. Pull-down assays [17] were run on SDS/PAGE and transferred on to PVDF membrane, then probed with anti-pY-HRP antibodies [17] and subsequently stained with Coomassie Blue to detect the NusA fusion. Quantification and statistical analysis of replica blots is shown.

We then directly tested the ability of the Tyr381 and Tyr409 motifs within the C-terminal region of Cbp (aa 352–424) to bind SFK SH2 domains (Figure 2B). All SFK SH2 domains could bind to these two pY motifs, with Lyn, Src, Hck and Lck displaying a significant relative preference for the pTyr409 site over the pTyr381 site within this pY-Y2H assay system.

To ascertain if this apparent preference for the pTyr409 motif by the SH2 domain of Lyn, determined via the pY-Y2H system, was due to affinity differences, we employed fluorescence spectroscopy with purified SH2 domains and fluorescently labelled phospho-peptides to directly measure their kinetic parameters (Figure 2C). The expressed SH2 domains were purified by pY-affinity chromatography, indicating that they were functional. The molecular mass for each SH2 domain was confirmed by MS (Lyn-SH2, 12300.57 Da; Src-SH2, 12285.89 Da; and Hck-SH2, 12223.55 Da). These values are all within 1 Da of the respective theoretical masses, based on the expected amino acid sequences. The correct folding of each SH2 domain was confirmed by their CD spectra (Supplementary Figure S1a available at http://www.BiochemJ.org/bj/442/bj4420611add.htm). The CD spectra overlay well and are consistent with the mixed α–β SH2 fold, as seen previously for other SFK SH2 domains [33,34]. The thermal stability of each protein was assessed by monitoring their CD ellipticity as a function of temperature (Supplementary Figures S1b–S1d). The thermal denaturation of Lyn-SH2 and Hck-SH2 was reversible and fitted well to a two-state model, whereas Src-SH2 precipitated upon unfolding. All three SH2 domains appeared to have similar thermal stability: Lyn-SH2, Tm=64.6±0.4°C; Hck-SH2, Tm=62.1±0.5°C; and Src-SH2, Tmapp~58°C. These Tm values are in good agreement with the published values for SH2 domains [35,36]. Taken together, these data indicate that the E. coli expressed SH2 domains used in this study were intact, folded, functional and stable.

The affinity of each SH2 domain for the FITC–HMTA, FITC–Cbp381 and FITC–Cbp409 phospho-tyrosyl peptides was measured using fluorescence spectroscopy (Figure 2C and Table 1, and Supplementary Figure S2 available at http://www.BiochemJ.org/bj/442/bj4420611add.htm). The HMTA sequence (EPQpYEEIPIYL), also called the ‘pYEEI’ peptide, is a high-affinity Src-family SH2-binding sequence [37]. The measured affinities of the three SH2 domains for the HMTA peptide agree well with the published sub-micromolar Kd values for isolated SFK SH2 domains (Kd values 0.1–0.9 μM) [3840]. All three SH2 domains bound the Cbp409 peptide with an affinity that was comparable with their affinity for the HMTA peptide. However, the affinity of each SH2 domain for the Cbp381 peptide was considerably weaker (~7-fold) than their affinity for the HMTA peptide.

Table 1
Affinity of Cbp/HMTA pY-peptides for the SH2 domains of Lyn, Hck and Src

Kd values are the mean from three separate titrations and the uncertainties represent the propagated standard errors from the fits. The value in parentheses indicates the fold-change between the Kd values for the HMTA peptide and the respective Cbp peptide.

Protein Kd FITC–HMTA (μM) Kd FITC–Cbp409 (μM) Kd FITC–Cbp381 (μM) 
Lyn-SH2 0.58±0.27 0.97±0.23 (1.68) 4.18±0.74 (7.19) 
Hck-SH2 0.65±0.28 0.60±0.16 (0.93) 4.96±2.03 (7.63) 
Src-SH2 0.31±0.11 0.67±0.15 (2.20) 2.21±0.47 (7.20) 
Protein Kd FITC–HMTA (μM) Kd FITC–Cbp409 (μM) Kd FITC–Cbp381 (μM) 
Lyn-SH2 0.58±0.27 0.97±0.23 (1.68) 4.18±0.74 (7.19) 
Hck-SH2 0.65±0.28 0.60±0.16 (0.93) 4.96±2.03 (7.63) 
Src-SH2 0.31±0.11 0.67±0.15 (2.20) 2.21±0.47 (7.20) 

We then sought to assay the ability of Lyn, Src and Hck SH2 domains to preferentially interact with phosphorylated Cbp within cell lysates by performing pull-down assays (Figure 2D). These results suggest that Lyn has a significant preference for phospho-Cbp compared with Src or Hck within the context of the phospho-protein milieu of a cell lysate. Taken together, these data demonstrate a high degree of specificity and affinity of the Tyr381, and in particular the Tyr409, motifs of Cbp for binding to the SH2 domain of Lyn.

Linking the Tyr314 and Tyr409 motifs of Cbp can mediate Csk binding and polyubiquitination of Lyn

Having previously established that the Tyr314 motif of Cbp interacts with the SH2 domains of Csk and SOCS1, mediating inactivation/down-regulation of Lyn [17], in the context of full-length Cbp, we looked at fusing this motif with the Tyr409 motif, having shown in the present study that it has the highest selectivity for Lyn (Figure 3). This was in the expectation that this construct could mediate endogenous Csk association with Lyn, as well as promote poly-ubiquitination of Lyn via endogenous SOCS1. In COS-7 cells, the isolated Tyr409 motif was indeed phosphorylated by wild-type and dominant active Lyn and interacted with full-length Lyn (Figure 3A). The Tyr314 motif of Cbp is only phosphorylated by hyper-active Lyn in COS-7 cells as an isolated eGFP fusion, but does not bind full-length Lyn in co-immunoprecipitation assays as does the Tyr409 motif, confirming our pY-Y2H data (Figure 3B) and previous findings [17,19]. Adding the Tyr409 motif to the eGFP–Tyr314 fusion significantly enhances its level of phosphorylation by both wild-type and hyper-active Lyn, potentially due to the synergism via the pTyr409 motif binding active Lyn promoting phosphorylation of the Tyr314 motif. This Tyr314–Tyr409 fusion also displayed significant association with endogenous Csk, presumably due to high level Tyr314 phosphorylation (Figure 3C). This construct encompassing just the Tyr314 and Tyr409 motifs of Cbp is thus able to replicate the main functions of full-length Cbp, namely phosphorylation by a SFK (Lyn), interaction with an SFK (Lyn) and binding of Csk. We then tested the ability of this fusion to mediate the other notable function of the Tyr314 motif, i.e. SOCS1-directed polyubiquitination of Lyn [17]. Indeed, the combination of the Tyr314 and Tyr409 motifs elevated the level of polyubiquitination of Lyn, particularly with constitutively active LynY508F, commensurate with the induction of endogenous SOCS1 that the hyper-active Lyn mediates (Figure 3D). This shows that these two motifs (Tyr314 and Tyr409) can, independently of the context of the full-length Cbp molecule, mediate their known functions, i.e. be phosphorylated by Lyn and co-ordinate interaction with Lyn (Tyr409) and Csk/SOCS1 (Tyr314). This interaction can result in enhanced phosphorylation of the C-terminal negative tyrosine residue [17] and increased poly-ubiquitination of active Lyn (Figure 3D). Furthermore, we also show that SOCS1 (FLAG-tagged) was able to co-immunoprecipitate with the Tyr314/Tyr409 but not the Phe314/Phe409 eGFP fusion construct, and that the co-immunoprecipitation was strongest when hyper-active Lyn (Y508F) was co-expressed (Figure 3E).

Fusion of the Lyn-binding Tyr409 Cbp motif with the Csk/SOCS1-binding Tyr314 motif leads to increased Csk binding and polyubiquitination of Lyn

Figure 3
Fusion of the Lyn-binding Tyr409 Cbp motif with the Csk/SOCS1-binding Tyr314 motif leads to increased Csk binding and polyubiquitination of Lyn

(A) The Tyr409 motif of Cbp is phosphorylated by wild-type and hyper-active Lyn and interacts with Lyn in COS-7 cells. COS-7 cells transfected with the indicated plasmids were lysed in raft buffer and immunoprecipitated with anti-eGFP and/or Lyn antibodies as described in the Materials and methods section. Western blots of the immunoprecipitates (IP) and total protein lysates were performed using anti-Lyn, anti-eGFP and anti-pY-HRP antibodies. (B) The Tyr314 motif of Cbp is phosphorylated by active Lyn but does not bind Lyn. COS-7 cells transfected with the constructs as indicated were analysed by immunoprecipitation and Western blotting as described in (A). (C) An eGFP fusion combining the Tyr314 and Tyr409 motifs of Cbp is phosphorylated by Lyn and binds to Csk. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-eGFP or anti-Csk (Santa Cruz Biotechnology) antibodies. Western blots of immunoprecipitates and total protein lysates were probed with anti-Csk, anti-eGFP and anti-pY-HRP antibodies. (D) An eGFP fusion combining the Tyr314 and Tyr409 motifs of Cbp enhances the polyubiquitination of hyper-active Lyn (Y508F). COS-7 cells transfected with the indicated plasmids were lysed, immunoprecipitated and analysed by Western blotting as described in (A). (E) SOCS1 co-immunoprecipitates with eGFP–Tyr314/Tyr409, but not eGFP–Phe314/Phe409, and enhanced with co-expression of hyperactive Lyn (Y508F). COS-7 cells transfected with the indicated plasmids were lysed, immunoprecipitated and analysed by Western blotting as described in (A). Ub, ubiquitin; WT, wild-type.

Figure 3
Fusion of the Lyn-binding Tyr409 Cbp motif with the Csk/SOCS1-binding Tyr314 motif leads to increased Csk binding and polyubiquitination of Lyn

(A) The Tyr409 motif of Cbp is phosphorylated by wild-type and hyper-active Lyn and interacts with Lyn in COS-7 cells. COS-7 cells transfected with the indicated plasmids were lysed in raft buffer and immunoprecipitated with anti-eGFP and/or Lyn antibodies as described in the Materials and methods section. Western blots of the immunoprecipitates (IP) and total protein lysates were performed using anti-Lyn, anti-eGFP and anti-pY-HRP antibodies. (B) The Tyr314 motif of Cbp is phosphorylated by active Lyn but does not bind Lyn. COS-7 cells transfected with the constructs as indicated were analysed by immunoprecipitation and Western blotting as described in (A). (C) An eGFP fusion combining the Tyr314 and Tyr409 motifs of Cbp is phosphorylated by Lyn and binds to Csk. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-eGFP or anti-Csk (Santa Cruz Biotechnology) antibodies. Western blots of immunoprecipitates and total protein lysates were probed with anti-Csk, anti-eGFP and anti-pY-HRP antibodies. (D) An eGFP fusion combining the Tyr314 and Tyr409 motifs of Cbp enhances the polyubiquitination of hyper-active Lyn (Y508F). COS-7 cells transfected with the indicated plasmids were lysed, immunoprecipitated and analysed by Western blotting as described in (A). (E) SOCS1 co-immunoprecipitates with eGFP–Tyr314/Tyr409, but not eGFP–Phe314/Phe409, and enhanced with co-expression of hyperactive Lyn (Y508F). COS-7 cells transfected with the indicated plasmids were lysed, immunoprecipitated and analysed by Western blotting as described in (A). Ub, ubiquitin; WT, wild-type.

The Tyr381 and Tyr409 motifs of Cbp are phosphorylated by and interact with Lyn

In an attempt to enhance the ability of our fusion to interact with Lyn and SFKs, as well as enhance their poly-ubiquitination/degradation, we decided to test the Tyr381 and Tyr409 motifs in combination with the SB of SOCS1. Theorizing that in combination the Tyr381/Tyr409 motifs would show enhanced phosphorylation by active Lyn and potentially interact with two Lyn molecules, whereas inclusion of the SB would circumvent the need of endogenous SOCS1 induction/expression to mediate polyubiquitination/degradation. We first tested the Tyr381/Tyr409 motif combination, encompassed by residues 352–424 of Cbp (Figure 4). In the present study, we found that the level of phosphorylation of the Tyr381/Tyr409 fusion correlated with the activity status of Lyn, i.e. no phosphorylation by inactive Lyn, moderate phosphorylation by wild-type Lyn and strong phosphorylation by hyperactive Lyn (Figure 4A). Furthermore, this fusion interacted strongly with Lyn, as assayed by co-immunoprecipitation experiments (Figure 4B). We then tested the ability of the Tyr381/Tyr409 fusion to be phosphorylated by and interact with activated endogenous Lyn. Significantly, this fusion was readily phosphorylated by activated (using vanadate) endogenous Lyn as well as interacting strongly with Lyn as assayed by co-immunoprecipitation (Figure 4C).

An eGFP fusion combining the Tyr381 and Tyr409 motifs of Cbp senses the activity status of Lyn and interacts with endogenous activated Lyn

Figure 4
An eGFP fusion combining the Tyr381 and Tyr409 motifs of Cbp senses the activity status of Lyn and interacts with endogenous activated Lyn

(A) The two C-terminal tyrosine residues of Cbp (Tyr381 and Tyr409) are phosphorylated by Lyn, relative to the activity status of Lyn. COS-7 cells were transfected with the constructs indicated, were lysed and immunoprecipitated essentially as previously described using raft buffer [17]. eGFP was immunoprecipitated using anti-eGFP antibodies (Santa Cruz Biotechnology). Immunoprecipitates (IP) and total protein lysates were run on SDS/PAGE gels, transferred to PVDF membrane and probed with the following antibodies; anti-pY-HRP (Millipore), anti-eGFP, anti-Lyn (Santa Cruz Biotechnology) and anti-β-actin (Abcam). (B) The phosphorylated Tyr381/Tyr409 region of Cbp co-immunoprecipitates with Lyn. Lysates of COS-7 cells transfected with the indicated plasmids were immununoprecipitated with anti-eGFP or anti-Lyn antibodies as described in (A). Western blots were probed with anti-pY-HRP, anti-eGFP and anti-Lyn antibodies. (C) Interaction of the Tyr381/Tyr409 motifs of Cbp with endogenous Lyn. COS-7 cells transfected with the indicated plasmids were serum starved for 16 h, then stimulated with or without NaVO4 (10 mM) for 30 min, as indicated, prior to lysis in raft buffer. Lysates were immunoprecipitated with anti-Lyn antibodies or anti-eGFP antibodies and analysed by Western blotting as described in (A). WT, wild-type.

Figure 4
An eGFP fusion combining the Tyr381 and Tyr409 motifs of Cbp senses the activity status of Lyn and interacts with endogenous activated Lyn

(A) The two C-terminal tyrosine residues of Cbp (Tyr381 and Tyr409) are phosphorylated by Lyn, relative to the activity status of Lyn. COS-7 cells were transfected with the constructs indicated, were lysed and immunoprecipitated essentially as previously described using raft buffer [17]. eGFP was immunoprecipitated using anti-eGFP antibodies (Santa Cruz Biotechnology). Immunoprecipitates (IP) and total protein lysates were run on SDS/PAGE gels, transferred to PVDF membrane and probed with the following antibodies; anti-pY-HRP (Millipore), anti-eGFP, anti-Lyn (Santa Cruz Biotechnology) and anti-β-actin (Abcam). (B) The phosphorylated Tyr381/Tyr409 region of Cbp co-immunoprecipitates with Lyn. Lysates of COS-7 cells transfected with the indicated plasmids were immununoprecipitated with anti-eGFP or anti-Lyn antibodies as described in (A). Western blots were probed with anti-pY-HRP, anti-eGFP and anti-Lyn antibodies. (C) Interaction of the Tyr381/Tyr409 motifs of Cbp with endogenous Lyn. COS-7 cells transfected with the indicated plasmids were serum starved for 16 h, then stimulated with or without NaVO4 (10 mM) for 30 min, as indicated, prior to lysis in raft buffer. Lysates were immunoprecipitated with anti-Lyn antibodies or anti-eGFP antibodies and analysed by Western blotting as described in (A). WT, wild-type.

Fusing the SB of SOCS1 to the Tyr381/Tyr409 motifs of Cbp can induce polyubiquitination and degradation of Lyn

Taking the results described above, we then proceeded to test the addition of the SB of SOCS1 for its ability to mediate poly-ubiquitination and proteasomal degradation of Lyn. We tested this construct in COS-7 cells and found the addition of the SB to the Tyr381/Tyr409 region could indeed mediate significant polyubiquitination of Lyn that was dependent upon the level of activity of the kinase and required the presence of the Lyn-binding Tyr381/Tyr409 motifs (Figure 5A). This was best revealed in the presence of the proteasomal inhibitor MG132 (Figure 5A). Also, in ubiquitin (HA-tagged) immunoprecipitates (Figure 5B), we could detect increased levels of polyubiqitinated Lyn, as well as total polyubiquitinated proteins, when the Tyr381/Tyr409-SB construct was co-expressed, compared with those expressing the Phe381/Phe409-SB construct. Indeed, we could still detect enhanced ubiquitination of wild-type Lyn in the absence of MG132 (Figure 5C). Furthermore, the lower levels of Lyn seen when the SB was added to the Tyr381/Tyr409 fusion could be reversed in the presence of MG132 (Figure 5D). Significant ubiquitination of Lyn was not seen when the SB or Tyr381/Tyr409 motifs were used alone, or when the tyrosine motifs were mutated to phenylalanine in the construct containing the Tyr381/Tyr409 motifs and the SB (Figures 5A, 5C and 5D). In addition, the Tyr381/Tyr409-SB construct was relatively stable, compared with the Phe381/Phe409-SB or SB alone eGFP fusions in COS-7 cells, and could mediate a reduced level of overexpressed wild-type Lyn (Figure 5E). These results illustrate that fusing the high-affinity Lyn-binding pY motifs of Cbp with the SB of SOCS1 generates a fusion protein that can direct the polyubiquitination and proteasomal degradation of kinase-active Lyn in COS-7 cells.

Fusion of the Lyn binding Tyr381/Tyr409 Cbp motifs with the SB of SOCS1 leads to increased polyubiquitination and proteasomal degradation of Lyn

Figure 5
Fusion of the Lyn binding Tyr381/Tyr409 Cbp motifs with the SB of SOCS1 leads to increased polyubiquitination and proteasomal degradation of Lyn

(A) Linking the SB of SOCS1 to the Tyr381/Tyr409 motifs of Cbp promotes the polyubiquitination of Lyn, dependent upon its kinase activity. COS-7 cells transfected with the indicated constructs, including an HA-tagged ubiquitin-expressing plasmid (HA-Ub) [17] were incubated with MG132 (40 μM) for 12 h before being lysed and immunoprecipitated (IP) with anti-eGFP or anti-Lyn antibodies. Western blots were probed with anti-Lyn, anti-HA (Millipore), anti-eGFP and anti-pY-HRP antibodies. (B) Immunoprecipitating ubiquitinated proteins shows increased ubiquitinated Lyn from cells expressing the Tyr381/Tyr409-SB construct compared with the Phe381/Phe409-SB construct. COS-7 cells transfected with the indicated constructs were incubated with MG132 (40 μM) for 12 h before being lysed and immunoprecipitated with anti-HA (ubiquitin-HA) antibodies. Western blots of HA immunoprecipitates were probed with anti-Lyn (upper panel) and anti-HA (ubiquitin, lower panel). Lysates were probed with anti-eGFP and anti-Lyn antibodies. (C) The ubiquitination of Lyn promoted by the Tyr381/Tyr409-SB construct requires both the tyrosine motifs and SB elements. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-Lyn antibodies. Western blots were probed with anti-Lyn and anti-HA antibodies. (D) The Tyr381/Tyr409-SB constructs promotion of decreased Lyn levels was mitigated by the addition of the proteasomal inhibitor MG132. COS-7 cells transfected with the indicated constructs were incubated with or without MG132 (40 μM) for 12 h before being lysed. Western blots of equal amounts of total protein were probed with anti-Lyn antibodies. (E) The Tyr381/Tyr409-SB construct causes a decrease in Lyn expression relative to β-actin. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-eGFP antibodies. Western blots of immunoprecipitates and total protein lysates were probed with anti-Lyn, anti-eGFP, and anti-pY-HRP and anti-β-actin (Abcam) antibodies. WT, wild-type.

Figure 5
Fusion of the Lyn binding Tyr381/Tyr409 Cbp motifs with the SB of SOCS1 leads to increased polyubiquitination and proteasomal degradation of Lyn

(A) Linking the SB of SOCS1 to the Tyr381/Tyr409 motifs of Cbp promotes the polyubiquitination of Lyn, dependent upon its kinase activity. COS-7 cells transfected with the indicated constructs, including an HA-tagged ubiquitin-expressing plasmid (HA-Ub) [17] were incubated with MG132 (40 μM) for 12 h before being lysed and immunoprecipitated (IP) with anti-eGFP or anti-Lyn antibodies. Western blots were probed with anti-Lyn, anti-HA (Millipore), anti-eGFP and anti-pY-HRP antibodies. (B) Immunoprecipitating ubiquitinated proteins shows increased ubiquitinated Lyn from cells expressing the Tyr381/Tyr409-SB construct compared with the Phe381/Phe409-SB construct. COS-7 cells transfected with the indicated constructs were incubated with MG132 (40 μM) for 12 h before being lysed and immunoprecipitated with anti-HA (ubiquitin-HA) antibodies. Western blots of HA immunoprecipitates were probed with anti-Lyn (upper panel) and anti-HA (ubiquitin, lower panel). Lysates were probed with anti-eGFP and anti-Lyn antibodies. (C) The ubiquitination of Lyn promoted by the Tyr381/Tyr409-SB construct requires both the tyrosine motifs and SB elements. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-Lyn antibodies. Western blots were probed with anti-Lyn and anti-HA antibodies. (D) The Tyr381/Tyr409-SB constructs promotion of decreased Lyn levels was mitigated by the addition of the proteasomal inhibitor MG132. COS-7 cells transfected with the indicated constructs were incubated with or without MG132 (40 μM) for 12 h before being lysed. Western blots of equal amounts of total protein were probed with anti-Lyn antibodies. (E) The Tyr381/Tyr409-SB construct causes a decrease in Lyn expression relative to β-actin. COS-7 cells transfected with the indicated plasmids were lysed and immunoprecipitated with anti-eGFP antibodies. Western blots of immunoprecipitates and total protein lysates were probed with anti-Lyn, anti-eGFP, and anti-pY-HRP and anti-β-actin (Abcam) antibodies. WT, wild-type.

Effect of the Lyn-binding motif – SB fusion on cancer cell lines

Next, we sought an understanding of the effect of expressing the Tyr381/Tyr409-SB construct in cancer cell lines (MCF-7, LNCaP and DU145) that are known to utilize Lyn/SFKs for important signal transduction events [12,41,42]. These breast (MCF-7) and prostate (LNCaP and DU145) cell lines have been reported to express active Lyn kinase and utilize this enzyme for important cell signalling events [11,12,42]. We observed that DU145 cells have high levels of Lyn kinase activity, LNCaP cells have a moderate level, whereas MCF7 cells have very low levels of active Lyn kinase (Figure 6A). Indeed, MCF-7 cells have very low levels of total Lyn protein, compared with DU145 and LNCaP cells (Figure 6B). Two isoforms of Lyn exist (p53 and p56) due to alternative splicing, and DU145 cells appear to express more of the p53 isoform, whereas LNCaP cells have a predominance of the p56 isoform (Figure 6B). Further, the three cell lines expressed Cbp and SOCS1, suggesting the endogenous Lyn/Cbp/SOCS1 pathway exists within these cells (Figure 6B). Significantly, transient expression of the Tyr381/Tyr409-SB fusion in DU145 cells had a significant ability to decrease the level of Lyn commensurate with loss of caspase 3 (Figure 6C). Such effects were not observed in LNCaP or MCF-7 cells expressing the same construct (results not shown). Furthermore, we were unable to derive stable clones of DU145 expressing the Tyr381/Tyr409-SB fusion and transfected cells undergoing selection displayed a classic rounded apoptotic phenotype with membrane blebbing, whereas Phe381/Phe409-SB could be tolerated and these cells retained their normal phenotype (Figure 6D). LNCaP and MCF-7 cells could both stably express the Tyr381/Tyr409-SB and Phe381/Phe409-SB fusions with no significant morphological changes (Figure 6D).

Expression of the Tyr381/Tyr409-SB construct in the Lyn-expressing cancer cell line DU145 promotes Lyn degradation and reduced caspase 3 levels, and is not compatible with cell expansion/survival

Figure 6
Expression of the Tyr381/Tyr409-SB construct in the Lyn-expressing cancer cell line DU145 promotes Lyn degradation and reduced caspase 3 levels, and is not compatible with cell expansion/survival

(A) DU145 cells have high levels of Lyn activity. Lyn was immunoprecipitated (IP) from DU145, LNCaP and MCF-7 cells and then used in in vitro kinase assays using bacterially expressed and purified GST (glutathione transferase)–Cbp (G-Cbp) as an exogenous substrate. Kinase assays were stopped at various time points by the addition of SDS sample buffer and heat denatured. Samples were run on SDS/PAGE and transferred on to PVDF membrane, then probed with anti-pY-HRP, anti-GST and anti-Lyn antibodies. (B) Lyn, Cbp and SOCS1 expression in cancer cell lines DU145, LNCaP and MCF-7. Lysates of cells were run on SDS/PAGE gels and transferred on to PVDF membrane, then probed with anti-Lyn, anti-Cbp, anti-SOCS1 and anti-β-actin antibodies. Short and long exposures of anti-Lyn Western blots are shown to depict the low-level expression of Lyn in MCF-7 cells. (C) Transient expression of the Tyr381/Tyr409-SB eGFP fusion in DU145 cells decreases Lyn expression and lower caspase 3 levels. DU145 cells were transiently transfected with the indicated constructs and lysates prepared 48 h post-transfection. Samples were run on SDS/PAGE and transferred to PVDF membrane before being probed with anti-Lyn, anti-caspase 3 (Cell Signaling Technology), anti-eGFP and anti-β-actin antibodies. (D) Expression of the Tyr381/Tyr409-SB construct in DU145, but not LNCaP or MCF-7 cells, promotes apparent apoptotic cell morphology and is not compatible with cell expansion. Cells were transfected with the linearized constructs indicated and after 48 h cells were selected for integrated plasmids with G418 (1 mg/ml) for 7 days. Surviving G418-resistant cells were then examined for eGFP expression by fluorescence microscopy (IF) as well as phase contrast (PC) at ×20 magnification on an Olympus IX71 microscope using an Olympus DP70 camera. Close-up images of the PC image of cells expressing the eGFP constructs are shown on the right (PC enlarged).

Figure 6
Expression of the Tyr381/Tyr409-SB construct in the Lyn-expressing cancer cell line DU145 promotes Lyn degradation and reduced caspase 3 levels, and is not compatible with cell expansion/survival

(A) DU145 cells have high levels of Lyn activity. Lyn was immunoprecipitated (IP) from DU145, LNCaP and MCF-7 cells and then used in in vitro kinase assays using bacterially expressed and purified GST (glutathione transferase)–Cbp (G-Cbp) as an exogenous substrate. Kinase assays were stopped at various time points by the addition of SDS sample buffer and heat denatured. Samples were run on SDS/PAGE and transferred on to PVDF membrane, then probed with anti-pY-HRP, anti-GST and anti-Lyn antibodies. (B) Lyn, Cbp and SOCS1 expression in cancer cell lines DU145, LNCaP and MCF-7. Lysates of cells were run on SDS/PAGE gels and transferred on to PVDF membrane, then probed with anti-Lyn, anti-Cbp, anti-SOCS1 and anti-β-actin antibodies. Short and long exposures of anti-Lyn Western blots are shown to depict the low-level expression of Lyn in MCF-7 cells. (C) Transient expression of the Tyr381/Tyr409-SB eGFP fusion in DU145 cells decreases Lyn expression and lower caspase 3 levels. DU145 cells were transiently transfected with the indicated constructs and lysates prepared 48 h post-transfection. Samples were run on SDS/PAGE and transferred to PVDF membrane before being probed with anti-Lyn, anti-caspase 3 (Cell Signaling Technology), anti-eGFP and anti-β-actin antibodies. (D) Expression of the Tyr381/Tyr409-SB construct in DU145, but not LNCaP or MCF-7 cells, promotes apparent apoptotic cell morphology and is not compatible with cell expansion. Cells were transfected with the linearized constructs indicated and after 48 h cells were selected for integrated plasmids with G418 (1 mg/ml) for 7 days. Surviving G418-resistant cells were then examined for eGFP expression by fluorescence microscopy (IF) as well as phase contrast (PC) at ×20 magnification on an Olympus IX71 microscope using an Olympus DP70 camera. Close-up images of the PC image of cells expressing the eGFP constructs are shown on the right (PC enlarged).

DISCUSSION

SFKs, and in particular Lyn, are becoming an increasingly important potential therapeutic target for several leukaemias and cancers, including prostate cancer [12,4245]. In the present study, we have utilized our knowledge of a Lyn/SFK-regulating pathway that involves Cbp as a scaffold protein to bring together negative regulators Csk and SOCS1 with Lyn/SFK [19], in the pursuit of developing minimal fusion proteins able to enhance this down-regulation of Lyn/SFK. In the present study, we have developed a protein construct consisting of the Lyn SH2-domain-binding pY motifs from Cbp and the SB of SOCS1, which down-regulates Lyn in overexpression systems, as well as in the prostate cancer cell line DU145, which has high Lyn activity levels and results in reduced survival of this prostate cancer cell line when stably expressed. Potentially, this offers an additional avenue to the development of anti-prostate cancer treatments.

We were able to demonstrate that the pY motifs Tyr381 and Tyr409 from Cbp, which have homology to SFK SH2-domain-binding pY consensus sequences [14,37] functioned as good substrates for active Lyn and could bind to its SH2 domain with high specificity and affinity (Kd=0.97±0.23 μM) when used in isolation, as well as in the full-length molecule, with the Tyr409 motif having a ~7-fold higher affinity than the Tyr381 motif. The addition of the Tyr409 site to another pY motif that binds Csk/SOCS1 enhanced the phosphorylation of this additional (Tyr314) site and allowed significant interaction of the fusion with its partner proteins Csk and SOCS1.

Adding the SB of SOCS1 to the Lyn-binding pY motifs directed the ubiquitination machinery that interacts with the SB through the elongin B/C complex to enhance the polyubiquitination and proteasomal degradation of Lyn. Having thus established the ability of this fusion between Cbp and SOCS1 in overexpression systems, we then turned to assessing its potential to down-regulate Lyn in a cancer cell line that has high Lyn activity (Figure 6) and has previously been shown to utilize this molecule for essential cellular signalling, namely the prostate cancer cell line DU145 [12,41,42]. When transiently transfected into DU145 cells, the eGFP–Tyr381/Tyr409-SB chimaera significantly reduced Lyn levels, as well as causing reduced caspase 3 levels, suggesting the reduced Lyn was inducing the apoptotic pathway. These cells also displayed a rounded phenotype with membrane blebbing typical of apoptotic cells. Interestingly, attempts to generate stable expression of the eGFP–Tyr381/Tyr409-SB fusion in DU145 cells failed, whereas the control construct with the tyrosine residues mutated to phenylalanine could be maintained. These results are suggestive that Lyn is required for expansion of this prostate cancer cell line, and indeed this is consistent with previous studies that targeted Lyn in these cells [12,42].

Taken together, these studies illustrate how our established knowledge on the regulation of Lyn/SFK levels and its enzymatic activity [19] can be used for the development of new molecules, through the fusing of functional motifs/domains from different proteins within a signal transduction pathway, which can promote the degradation of Lyn in cells and appears to inhibit cancer cell line expansion. Furthermore, this construct could be used to help dissect the role of Lyn in different cells/tissues through its ability to down-regulate active Lyn. However, its ability to show preferential interaction with a subset of SFKs, predominantly Lyn, Hck and Src, and to a lesser extent Lck, could lessen its ability to be specific for Lyn, but may also allow a degree of differentiation with other members of the SFK family that have less specificity for this molecule. This may also assist in differentiating functions of SFK members that small molecule inhibitors, such as PP2, are unable to delineate due to them inhibiting all SFKs with similar specificities. Engineering of the pY motifs may allow the development of more specific interactions with individual SFK members, or indeed with other SH2-containing proteins of interest. These studies also illustrate the utility of the SB when fused to a targeting motif to direct degradation of specific molecules. Potentially, other specific targeting motifs could be fused to the SB, or indeed mimetics of the SB, to direct degradation of desired proteins within a cell.

Abbreviations

     
  • AML

    acute myeloid leukaemia

  •  
  • BCR

    breakpoint cluster region

  •  
  • Cbp

    Csk-binding protein

  •  
  • CML

    chronic myeloid leukaemia

  •  
  • Csk

    C-terminal Src kinase

  •  
  • DTT

    dithiothreitol

  •  
  • eGFP

    enhanced green fluorescent protein

  •  
  • HA

    haemagglutinin

  •  
  • HRP

    horseradish peroxidase

  •  
  • pY

    phospho-tyrosine

  •  
  • SH

    Src homology

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • SFK

    Src-family kinase

  •  
  • SOCS

    suppressor of cytokine signalling

  •  
  • SB

    SOCS box

  •  
  • Y2H

    yeast two-hybrid

AUTHOR CONTRIBUTION

Evan Ingley and Terrence D. Mulhern developed concepts, and designed and supervised experiments. Theresa Qiu, Sevgi Irtegun and Natalie Gunn prepared the SH2 domains and performed the fluorescence anisotropy experiments. Evan Ingley performed the pY Y2H experiments. Rhiannon Whiting, Christine Payne, Jiulia Satiaputra and Nicole Kucera performed experiments. Evan Ingley and Terrence Mulhern wrote the manuscript.

We thank Therese Weldt for technical assistance.

FUNDING

This work was supported by grants from the National Health and Medical Research Council [grant numbers 303101, 403987, 509115, 513714 and 634352], the Cancer Council of Western Australia and the Medical Research Foundation of Royal Perth Hospital.

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Supplementary data