The unique N-terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1

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1 The unique N-terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1 Raghuveera K. Goel, Sayem Miah, Kristin Black, Natasha Kalra, Chenlu Dai and Kiven E. Lukong Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Canada Keywords breast cancer; BRK; ; FRK; SRMS Correspondence K. E. Lukong, Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon S7N 5E5, Canada Fax: Tel: kiven.lukong@usask.ca (Received 6 March 2013, revised 20 June 2013, accepted 25 June 2013) doi: /febs SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS. Structured digital abstract SRMS physically interacts with Dok-1 by pull down (View Interaction: 1, 2) Dok-1 physically interacts with SRMS by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3) SRMS phosphorylates Dok-1 by protein kinase assay (View interaction) Dok-1 physically interacts with SRMS by anti tag coimmunoprecipitation (View interaction) Abbreviations BRK, breast tumour kinase; DAB, 3,3 -diaminobenzidine tetrahydrochloride; DAPI, 4,6-diamidino-2-phenylindole;, downstream of tyrosine kinases 1/docking protein 1; FRK, Fyn-related kinase; GFP, green fluorescent protein;, glutathione S-transferase; HEK 293, human embryogenic kidney 293; IHC, immunohistochemistry; PEI, polyethyleneimine; PTK, protein tyrosine kinase; PDN-SRMS, partial N-terminus deletion SRMS; SH2, Src-homology 2; SH3, Src-homology 3; SRMS, Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites; DN-SRMS, N-terminus deletion SRMS. FEBS Journal 2 (2013) ª 2013 FEBS 4539

2 N-terminus of SRMS and catalytic activity R. K. Goel et al. Introduction Protein tyrosine kinases (PTKs) comprise a distinct cohort of enzymes that function to phosphorylate the tyrosine residues on other proteins or, alternatively, those that lie within their own sequences, by autophosphorylation [1,2]. Deregulated activities of tyrosine kinases have been associated with many diseases, notably cancer. The Src family kinases comprise a prominent class of nonreceptor tyrosine kinases, whose family members include Src, Yes, Fyn, Lck, Hck, Blk and Lyn [3,4]. SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites), also known as PTK70, is a 488 amino acid nonreceptor tyrosine-kinase whose cellular role is unknown. SRMS (pronounced shrims ) is a member of a small family of intracellular Src-related tyrosine kinases, including breast tumour kinase (BRK) or PTK6 and Fyn-related kinase (FRK) or PTK5, called the FRK/PTK6 family [5]. The FRK/PTK6 family is distantly related to the Src-family, although members of this family share an exon intron structure distinct from the well-characterized Src family of tyrosine kinases [5]. Nonetheless, similar to Src family kinases, the functional structure of the FRK/PTK6 family proteins comprises one Src homology 3 (SH3) domain, one Src homology 2 (SH2) domain and one protein kinase domain [5]. Unlike PTK6 and FRK and other Src family kinases, SRMS is an understudied tyrosine kinase and its biochemical function, as well as its cellular and physiological roles, remains poorly characterized or unknown. SRMS was first cloned in 1994 by Kohmura et al. [6] from mouse embryonic neuroepithelial cells in an attempt to identify the genes that regulate the growth and differentiation of neuroepithelial cells [6]. The study ascribed SRMS expression to most mouse tissues, with the strongest expression levels being reported in the lung, testes and liver [6]. In addition, although SRMS was only poorly detected in embryonic day 11 brain tissues, an augmented and uniform expression of the transcript was observed in tissues derived from embryonic day 15 brain [6]. In 1997, Kawachi et al.[7] found that the SRMS transcript was highly expressed in the murine keratinocytes derived from the nontransformed epidermis sheet compared to tissues derived from melanomas or fibroblasts, suggesting an involvement in proliferation or differentiation of keratinocytes in the skin [7]. Interestingly, the human SRMS maps to chromosome 20q13.33, which is only 1.5 kbp upstream of the PTK6 gene, and this tight linkage not only complicates analysis of the PTK6 regulatory region, but also indicates that both genes may interact genetically [8]. The murine SRMS gene maps to chromosome 2 [6], which is syntenically conserved with the human chromosome 20q13.3, and therefore adjacent to the PTK6 gene in the same [9]. Interestingly, SRMS knockdown mice displayed no obvious phenotypical deviations, suggesting that the function of SRMS may be compensated for by other tyrosine kinases [6]. However, PTK6-null mice displayed an increased intestinal villus length and delayed enterocyte differentiation with a concomitant increase in intestinal epithelial cell proliferation and Akt activity [10]. This suggests that, although SRMS and PTK6 are genetically linked, they may discharge distinct cellular and physiological functions. Although PTK6 has been catalogued with over 20 characterized substrates and binding partners [8], no SRMS targets have been characterized to date. However, Takeda et al. [11] have described a proteomics study that revealed for the first time the adapter protein docking protein 1 (/p62) as a potential substrate of SRMS. is a scaffolding protein that functions to mediate protein protein interactions and has been characterized as a substrate of several tyrosine kinases, including the Src family kinases [12 16]. There are several uninvestigated facets regarding the physiological properties of SRMS, including its expression, activity, regulation and substrate specificity. PTK6 and FRK display opposing expression patterns and appear to exert contrasting functions in breast cancers [8,17], yet the expression pattern and function of SRMS in breast cancers is unknown. Because SRMS lacks the C-terminal autoinhibitory domain, a concise mechanism delineating the regulation of its catalytic activity cannot be predicted. In addition, unlike PTK6, the involvement of the SH3 and SH2 domains of SRMS in intramolecular interactions (i.e. towards autoinhibition) remains unclear. Similarly, the structure function correlation of the unique, extended N-terminal region of SRMS is unknown. The keys residues that contribute to the catalytic stability of SRMS, as well as the identification of the cellular substrates of SRMS, also remain to be probed. In the present study, we analyzed the expression of SRMS in breast cancer cell lines and tissues. We also generated a series of SRMS mutants aiming to study the regulation of SRMS localization, activity and substrate specificity. We identify the N-terminus of SRMS as an unforeseen essential element aiding in the perpetuation of enzymatic activity and we validate as a bona fide SRMS substrate. 45 FEBS Journal 2 (2013) ª 2013 FEBS

3 R. K. Goel et al. N-terminus of SRMS and catalytic activity Results The novel nonreceptor tyrosine kinase SRMS is overexpressed in breast carcinomas It was shown previously that the closest relative of SRMS, BRK, is overexpressed in the majority of breast cancer samples tested and has oncogenic properties [8,18 20]. The expression pattern of SRMS in the normal mammary epithelium or breast carcinomas or their derived cell lines has not been reported. We therefore aimed to determine SRMS expression in eight disparate breast cancer cell lines, namely, BT-20, MCF7, MDA-MB-231, MDA-MB-435, MDA-MB- 468, Au565, HBL-100 and SKBR3, and to compare its expression with that in a nontumourigenic mammary epithelial cell line, 184B5. We also compared the expression of SRMS with that of its highly characterized family member, BRK. The results obtained indicated a general overexpression of SRMS in six out of eight breast cancer cell lines, with HBL-100 exhibiting the highest levels of SRMS and MDA-MB-468 and AU565 displaying a lower expression (as demonstrated with a longer exposure of the X-ray film to the immunoblotted membrane) (Figs 1A and S1). Interestingly, we observed that SRMS was significantly downregulated in the nontumourigenic mammary epithelial cell line, 184B5. By contrast, BRK expression, although also observed in the majority, was found to clearly evade certain cell lines, namely MDA-MB-435, MDA- MB-468 and HBL-100. The data suggest that (a) SRMS, akin to BRK, is potentially overexpressed in the majority of breast cancer cell lines and (b) the expression of SRMS appears to evade the normal human breast cells at the same time as being significantly expressed in those derived from breast carcinomas. To corroborate these findings, we evaluated the expression of SRMS in human breast tissues and the normal mammary epithelium by immunohistochemistry (IHC). Tissue sections were derived from invasive ductal carcinomas of increasing pathological grade and the normal tissue sections were derived from adjacent sites. Representative images are shown in Fig. 1B. We detected a strong staining for SRMS in all carcinoma cases (Fig. 1B, a g) compared to normal tissue controls (Fig. 1B, i,j). SRMS intensity in the normal tissues ranged from +1 to +2. However, we observed intensities of +2 in grade 1 (Fig. 1B, a,b) and grade 2 (Fig. 1B, c,d) and +3 in grade 3 (Fig. 1B, e,g) invasive carcinoma samples. The specificity of the SRMS antibody was confirmed via staining with normal rabbit serum alone as a control (Fig. S2). Taken together, these findings suggest that the expression of SRMS is induced more in breast cancers compared to the normal mammary gland. Furthermore, both the breast carcinoma and the normal tissue samples exhibited cytoplasmic and nuclear staining of SRMS (Table S1). Details of the SRMS staining pattern across different pathological breast tissue samples are compiled in Table S1. A B a b Grade 1 Grade 2 Grade 3 Normal c e i d g j SRMS (short exposure) SRMS (long exposure) BRK β-tubulin Fig. 1. (A) SRMS is overexpressed in the human breast carcinomas. Lysates prepared from breast carcinoma cell lines and a cell line derived from the normal mammary epithelial tissue (184B5) were evaluated for SRMS expression via immunoblotting with antibodies against SRMS. b-tubulin was used as the loading control. (B) A representative image of SRMS expression via IHC on biological specimens procured from the breast cancer and normal adjacent tissues of female breast cancer patients. Tissue sections corresponding to (a) and (b) represent Grade 1; (c) and (d) represent Grade 2; and (e) and (g) represent Grade 3 breast carcinoma samples. (i, j) Adjacent normal breast tissues. Tissue sections were stained with anti-srms serum and specific binding was detected with ImmPRESS TM reagent followed by colour development in peroxidase substrate DAB. FEBS Journal 2 (2013) ª 2013 FEBS 4541

4 N-terminus of SRMS and catalytic activity R. K. Goel et al. SRMS localizes to punctate cytoplasmic structures Figure 1 shows that SRMS is highly expressed in breast cancer cell lines and tumours. The subcellular localization of SRMS is unknown and, in the absence of a putative myristoylation signal (dictating plasma-membrane localization) as found in Src [4] or a characteristic FRK-like nuclear localization signal (directing nuclear expression) [19], it was imperative to determine its unpredictable cellular localization pattern. To determine the true native and characteristic intracellular habitation of the protein, we began by pursuing an immunocytochemical analysis on endogenous SRMS in two breast cancer cell lines, MDA-MB-231 and AU565, and one cervical cancer cell line, HeLa. We found that endogenous SRMS localized to distinct punctate cytoplasmic structures in all three cell lines (Fig. 2A). Interestingly, the same pattern of localization was also detected in one other breast cancer cell line, SKBR3 (data not shown). No obvious nuclear colocalization with 4,6-diamidino-2-phenylindole (DAPI) (Fig. 2A, c, f, i) was observed, indicating that SRMS is predominantly a cytoplasmic protein. Next, we aimed to examine whether the endogenous localization pattern coincided with ectopically-expressed SRMS. A green fluorescent protein (GFP)-SRMS construct was used to transiently transfect the human embryogenic kidney 293 (HEK 293) cell line and localization was determined using fluorescence microscopy. The nuclei were stained with DAPI. Consistent with endogenous SRMS, overexpressed SRMS also produced a punctate localization pattern in the cytosol (Fig. 2B, a,c), whereas GFP alone was expressed diffusely throughout the cell (Fig. 2B, d,f). These findings suggest that SRMS is a cytoplasmic tyrosine kinase that localizes to specific punctate structures. Nevertheless, the localization of SRMS in sites of the cellular membrane was not abundantly clear from fluorescence microscopy analysis. To further substantiate these results and to specifically determine whether SRMS localizes to the membrane, a subcellular fractionation was employed in MDA-MB-231 and HeLa cell lines, including one other breast cancer cell line, HBL-100. Indeed, out of the three segregated cellular fractions, SRMS was found to be exclusive to the cytosolic fraction in all three cell lines, with no detectable levels determined in either the membrane or the nuclear fractions (Fig. 2C). Immunoblotting with antibodies against Sam68 and b-tubulin served as controls for the enrichment of nuclear and cytosolic/membrane proteins respectively. Therefore, using two different approaches (immunofluorescence microscopy and a subcellular fraction), we have demonstrated that SRMS has a cytosolic localization in breast cancer cells, as well as HeLa cervical cancer cells. The unique N-terminus region of SRMS regulates it tyrosine kinase activity Although SRMS belongs to the same family of nonreceptor tyrosine kinases as BRK and exhibits significant structural homology with this PTK, there are subtle variations in their overall primary amino acid sequence. First, SRMS lacks the C-terminal regulatory tail found in BRK and Src family kinases. Second, SRMS has an extended and unique N-terminus region, which is absent in other Src-related kinases (Fig. 3A). To understand the functional significance of this N-terminal region, to assess the enzymatic activity of SRMS and to identify key regulatory elements within the structure, we generated a series of GFP-tagged SRMS mutants. We constructed a SRMS mutant lacking the first 51 amino acids (DN-SRMS). We also generated K258M, Y342F and W223A mutants. These residues are conserved in BRK (K219, Y342 and W184) and Src-family kinases including, but not limited to, c-src (K298, Y419 and W263). Y342 and Y419 in BRK and c-src, respectively, lock the catalytic loop in the fullyactive conformation when autophosphorylated [21 23]. Previous studies have demonstrated that BRK is activated by autophosphorylation at Y342 because mutation of this site to alanine drastically reduced BRK activity [21]. K219 and K298 are critical for ATP binding and K219M and K298M mutants are kinase defective, dominant inhibitory forms of the enzymes [22 24]. The W184A mutant of BRK was shown to abolish kinase activity [22], whereas the analogous mutant in Src-family kinase, Hck, increased enzyme activity [25]. The enzymatic activity of wild-type SRMS and the contribution of the analogous residues in the regulation of SRMS activity have not been reported. To assess the overall activity of SRMS and its mutants, including DN-SRMS, K258M and W223A (Fig. 3A), we transiently transfected the GFP-tagged variants along with the wild-type protein in HEK 293 cells. For comparison, we also transfected the GFP-tagged BRK wild-type and two other mutants (kinase-dead BRK-K219M and constitutively active BRK-Y447F) and evaluated the total levels of tyrosine phosphorylation in cell lysates by immunoblotting using PY20, an antibody against phosphorylated tyrosines. First, we noted that wild-type SRMS exhibited strong intrinsic tyrosine kinase activity that is almost comparable to that of the wild-type BRK but not to 4542 FEBS Journal 2 (2013) ª 2013 FEBS

5 R. K. Goel et al. N-terminus of SRMS and catalytic activity A SRMS DAPI MERGE a b c MDA-231 d e f AU565 g h i HeLa Fig. 2. SRMS localizes to punctuate cytoplasmic structures in vivo. (A) Intracellular localization of endogenous SRMS was detected via indirect immunofluorecence in the MDA-MB-231 and AU565 human breast cancer cell lines, as well as the human cervical cancer cell line, HeLa. Immunoreactivity was visualized using primary anti-srms antibodies and fluorescein isothiocyanateconjugated secondary antibodies. Cells were counterstained with DAPI (blue). (B) HEK 293 cells were transiently transfected with plasmids encoding or GFP. The intracellular localization of exogenous GFP-tagged SRMS (a, c) and GFP alone (d, f) were detected via fluorescence microscopy. Cells were counterstained with DAPI (b, c, e, f). (C) Cells (from the indicated cell lines) were fractionated into the cytosolic, membrane and nuclear fractions and immunoblotted for the detection of SRMS. b-tubulin and Sam68 were used as controls for the cytosolic/membrane and nuclear compartments, respectively. SRMS is found in the cytosolic fraction of the indicated cell lines. B C GFP Cytosol GFP DAPI MERGE a b c d e f MDA-MB-231 Membrane Nucleus Cytosol HBL100 Membrane Nucleus Cytosol HeLa Membrane Nucleus SRMS Sam68 β-tubulin that of its hyperactive mutant BRK-Y447F (Fig. 3B). Second, and more significantly, we noted that deleting the 51 amino acid chain in the N-terminus region of SRMS (DN-SRMS) totally abolished kinase activity. Third, we observed an anticipated absence of kinase activity with the K258M mutant because abrogating the ATP-contacting site is expected to eliminate the intrinsic ability of the kinase to acquire and utilize the phosphate group derived from ATP for tyrosine phosphorylation. Because autophosphorylation of Src family kinases including BRK is a measure of enzyme activation [21,25], we aimed to examine whether the SRMS activity levels observed in Fig. 3B corresponded with the autophosphorylation of SRMS. We therefore FEBS Journal 2 (2013) ª 2013 FEBS 4543

6 N-terminus of SRMS and catalytic activity R. K. Goel et al. A SRMS 1 SH3 (51-112) SH2 ( ) Kinase ( ) 488 B GFP P N-SRMS 1 SH3 (51-112) SH2 ( ) Kinase ( ) 488 N-SRMS SH3-SRMS 1 SH3 (51-112) SH2 ( ) SH2 ( ) Kinase ( ) Kinase ( ) anti-gfp SH2-SRMS SRMS BRK 1 R147 K258 Y3 Residues W223 conserved in BRK and Src SH3 SH2 (120- Kinase P-rich (51-112) 212) ( ) P214 P226 Not conserved Residues P218 in BRK not conserved in and Src BRK,and Src 1 SH3 (51-112) SH3 (11-72) R105 SH2 (78-170) P175 P177 P179 W184 K219 Kinase ( ) Kinase ( ) Y Y anti-ptyr (short exposure) anti-ptyr (long exposure) C GFP: IP: anti-gfp GFP-BRK IgG GFP: IP: anti-gfp GFP-BRK D WT ΔN PΔN PA 2PA R147A ΔSH2 ΔSH3 W223A K258M anti-ptyr (Short Exposure) anti-ptyr (Long Exposure) 25 GFP anti-gfp anti-ptyr anti-gfp anti-srms Fig. 3. The N-terminus region of SRMS and its SH2 domain sustain a constitutively active form of the kinase. (A) Wild-type SRMS containing the globular functional domains; SH3, SH2 and the kinase domain. SRMS mutants; DN-SRMS (N-terminally deleted), PDN-SRMS (Partial N-terminus deletion), DSH3 (SH3 deletion), DSH2 (SH2 deletion) and its point mutated variants for the indicated amino acid residues are also shown. The critical regulatory residues W223A, K258 and Y3 are conserved with BRK and FRK. (B) Lysates prepared from HEK- 293 cells transfected with the indicated SRMS and BRK constructs were evaluated to determine the relative kinase activity of the variants using antibodies against total phosphotyrosines (bottom). Expression of the ectopic proteins was probed via anti-gfp (top). (C) Wild-type BRK and wild-type SRMS, as well as its indicated mutants, were immunoprecipitated from HEK 293 cell lysates and immunoblotted with anti-phosphotyrosine (top) to reveal autophosphorylation of the respective proteins. The immunoprecipitated proteins were probed via anti- GFP (bottom). SRMS auto-phosphorylation is strongly diminished in its Y3F mutant. (D) Transiently transfected variants of SRMS were analyzed for relative catalytic activity compared to wild-type SRMS via immunoblotting with phosphotyrosine antibodies (top). Anti-GFP was used to probe the ectopically-expressed proteins (middle). Anti-SRMS sera, targeting an epitope in the N-terminus region of SRMS, depict abrogated immunoreactivity with DN-SRMS and PDN-SRMS (bottom). analyzed the degree of autophosphorylation of wildtype SRMS and its three mutants, DN, W223A and Y3F. Y3 is the predicted autophosphorylation site within the kinase domain of SRMS. BRK wild-type was used as a positive control. The GFP-tagged constructs were transiently transfected into HEK 293 cells and the expressed proteins immunoprecipitated from the cell lysates using anti-gfp sera. The immunoprecipitates were then subjected to immunoblotting with anti-phosphotyrosine (PY20). As shown in Fig. 3C, although wild-type SRMS and BRK displayed prominent autophosphorylation, the SRMS-Y3F exhibited markedly reduced autophosphorylation, thereby conforming that this tyrosine residue is indeed the site for autophosphorylation in SRMS. Furthermore, with a longer exposure of the X-ray film, a subtle degree of autophosphorylation was noted in the W223A mutant, whereas DN-SRMS displayed null autophosphorylation (data not shown). We extended these studies to include additional SRMS mutants (Fig. 3D). The importance of the SH2 and SH3 domains in regulating the catalytic activity of 4544 FEBS Journal 2 (2013) ª 2013 FEBS

7 R. K. Goel et al. N-terminus of SRMS and catalytic activity BRK via intramolecular interactions has been demonstrated previously [20,21]. To determine the involvement of these globular domains in the regulation of the catalytic activity of SRMS, we generated GFPtagged SH3- and SH2-deleted mutants of SRMS (DSH3-SRMS and DSH2-SRMS, respectively). We also mutated a conserved arginine residue (R147) to alanine (R147A). This arginine residue resides in the SH2 domain and is part of the FLVRS motif that constitutes the phosphotyrosine recognition site in Src family kinases [26]. The analogous R175 in v-src makes contact with the phosphate group on tyrosine residues [26], and mutating this residue was shown to abrogate phosphotyrosine binding [27]. Furthermore, mutation of an analogous arginine (R105) in BRK resulted in reduced phosphorylation and association with its substrate STAP2 (signal transducing adaptor protein 2) [28]. Nonetheless, although DSH2 BRK mutant exhibits elevated kinase activity [25], the effect of the spatially disruptive R105 mutation on catalytic activity has not been reported. Therefore, we aimed to compare an analogous mutation in SRMS (R147) with its DSH2 mutant to evaluate catalytic repercussions. Figure 3B,C shows that the N-terminal region plays a critical role in stabilizing the intrinsic tyrosine kinase activity of SRMS. This N-terminus is replete with proline residues, where six such prolines, located proximal to the SH3 domain, were found to be closely stacked in a manner reflective of a discrete proline-rich motif. To assess its requirement wiith respect to SRMS kinase activity, we deleted this proline-rich segment to generate a partial N-terminal deletion mutant (PDN-SRMS). The SH2-kinase linker region of BRK contains proline residues that exhibit the PXXP SH3- binding motif and were also shown previously to form intramolecular interactions with the SH3 domain of BRK [22]. This linker region in SRMS contains three proline residues, albeit not conforming to a PXXP type motif (Fig. 3A). Therefore, we constructed two different mutants with knowledge of the spatial arrangement of the three prolines. One construct contained a single mutation of the proline (P214A and designated SRMS-PA) residing closest to the SH2 domain. The other two prolines, situated distally from the SH2 domain, were mutated together (P218A/ P226A and referred to as SRMS-2PA) in the second construct. All of these mutants, together with wildtype SRMS, DN-SRMS, SRMS-K258M and SRMS- W223A, were transiently transfected in HEK 293 cells to test the overall effects on kinase activity. Cell lysates were analyzed for immunoreactivity with antiphosphotyrosine sera to measure the relative kinase activity (Fig. 3D, top). The variants were generally expressed at comparable levels (Fig. 3D, middle and bottom). Of all the SRMS mutants examined, DSH2- SRMS (lane 7) displayed the lowest kinase activity compared to the wild-type SRMS, suggesting that the SH2 domain is essential for SRMS kinase activation. However, deletion of the SH3 domain did not significantly affect kinase activity (lane 8). P214A (lane 4) and 2PA (lane 5) mutations in the linker region also had little effect on kinase activity. Furthermore, the R147A mutation within the SH2 domain displayed kinase activity that was consistent with that of wildtype SRMS (compare lanes 1 and 6). Also, the PDN- SRMS failed to alter the catalytic activity of SRMS, suggesting that the short segment rich in proline residues does not display an exclusive role with respect to regulating SRMS tyrosine kinase activity. Taken together, our data demonstrate that the kinase activity of SRMS is regulated by its unique N-terminus sequence. Furthermore, by contrast to BRK [22], deletion of the SH2 domain of SRMS resulted in significantly reduced kinase activity. SRMS mutants display diverging subcellular localization patterns Figure 2A,B shows that both endogenous SRMS and ectopically-expressed localized to punctate cytoplasmic structures. The mechanisms regulating this punctate cytoplasmic localization are unknown. It is also not known whether this localization is dictated by intermolecular interactions with SRMS targets. It is conceivable, however, that the interaction of SRMS with certain endogenous cellular targets via SH3 and/ or SH2 domains might result in the retention of the protein kinase in specific cellular compartments. It is also possible that the unique, extended N-terminus region of SRMS regulates not only the activity of the enzyme, but also its subcellular localization via an unknown intermolecular interaction. We therefore tested the abilities of the N-terminal region, as well as the SH3 and SH2 domains, of SRMS to influence subcellular localization. Accordingly, full-length, as well as its GFP-tagged variants, DSH3-SRMS, DSH2-SRMS, DN-SRMS and SRMS- K258M, were expressed in HEK 293 cells and their localization was assessed by fluorescence microscopy (Fig. 4). As shown in Fig. 4 and consistent with Fig. 2B, wild-type SRMS localizes to punctate cytoplasmic structures in over 90% of the transfected cells. However, approximately 70% of the cells transfected with DN-SRMS exhibited a diffused localization, whereas the remainder displayed a punctate pattern of localization. Although almost % of the cells FEBS Journal 2 (2013) ª 2013 FEBS 4545

8 N-terminus of SRMS and catalytic activity R. K. Goel et al. DAPI MERGE DAPI MERGE WT SH2 SH3 N K258M Fig. 4. SRMS mutants exhibit divergent subcellular localization. GFP-tagged wild-type SRMS, as well as its mutants, DN-SRMS, K258M, DSH3 and DSH2, were transiently transfected in HEK 293 cells and localization was determined via fluorescence microscopy. DN-SRMS localization was found to be diffused in a major proportion of the transfected cell population. K258M exhibited a diffused cytoplasmic/ nucleo-cytoplasmic pattern. DSH2 displayed a predominantly diffused localization, whereas DSH3 revealed predominant punctate localization pattern. transfected with DSH3-SRMS localized to punctate cytoplasmic structures. DSH2-SRMS exhibited a diffused localization pattern in approximately 90% of transfected cells. Unexpectedly, SRMS-K258M displayed a cytoplasmic or nucleo-cytoplasmic diffused localization pattern in approximately % of transfected cells. Taken together, our data suggest that the N-terminal region of SRMS not only regulates the activity of the enzyme, but also can modulate the subcellular localization. Our findings also indicate that the SH2 domain of SRMS may contribute to sequester SRMS in specific cytoplasmic structures. is differentially expressed in breast cancer SRMS is a tyrosine kinase, although no SRMS substrate has been characterized to date. Recently, Takeda et al. [11] described a proteomics study that revealed potential substrates of the human SRMS protein for the first time. One of these potential substrates was the adapter protein,. has not been characterized as a SRMS substrate and/or binding partner. was first identified as an abundant tyrosine-hyperphosphorylated protein in chronic myelogenous leukaemia cells [29]. The scaffolding protein has also been validated as a substrate of various tyrosine kinases, including Src family kinase members [12,30]. is a reported tumour suppressor and has been shown to inhibit cell proliferation and leukaemogenesis, and to promote apoptosis [31,32]. The gene encoding this adapter protein is located in the human chromosome 2p13, a locus that is unstable in various human tumours [33]. Gene and protein expression studies have shown that is repressed in several human cancers, including head and neck cancer, lung, liver and gastric cancers, and Burkitt s lymphoma [34]. To our knowledge, the expression pattern of and interaction with its regulators in breast cancer cells has not been reported to date. Therefore, we analyzed expression in various breast cancer cell lines, as 4546 FEBS Journal 2 (2013) ª 2013 FEBS

9 R. K. Goel et al. N-terminus of SRMS and catalytic activity well as in the nontumourigenic breast epithelialderived 184B5, and correlated this with the expression of SRMS (Fig. 5A). We show that expression is low or absent in four cell lines, namely 184B5, MDA- MB-468, AU565 and SK-BR3 (Fig. 5A, lanes 6, 7 and 9). Three of these cell lines (184B5, MDA-MB-468 and AU565) also show decreased levels of SRMS. Interestingly, the elevated expression of in the other cell lines, especially in HBL-100, corresponded with the expression of SRMS (Fig. 5A, lane 8). Taken together, these data reveal a positive correlation between and SRMS expression in the breast cancer cells studied. Figure 2A,B shows that SRMS localizes to punctate cytoplasmic structures. Previous studies have shown that has a predominantly cytoplasmic/membrane localization [35], although the protein can also shuttle between the nucleus and the cytoplasm [15]. We next investigated whether SRMS and co-localized in vivo. Using antibodies specific to SRMS and, we found that SRMS and display different localization patterns (Fig. 5B). As with AU565 and MDA-MB-231, SRMS displays punctate cytoplasmic localization in HBL100 cells (Fig. 5B, a,d). Although is predominantly nuclear, minimal cytoplasmic A SRMS β-tubulin B a b c SRMS DAPI d e f SRMS/DAPI /DAPI SRMS/ g SRMS//DAPI Fig. 5. is differentially expressed in breast cancer cells. (A) expression was surveyed alongside SRMS in eight breast cancer cell lines, as well as the normal breast epithelial, 184B5, using antibodies against and SRMS, respectively. b-tubulin was used as the loading control. (B) Endogenous SRMS and co-localization was determined via indirect immunofluorescence in the HBL100 cell line using antibodies against SRMS (a) and (b). Cells were counterstained with DAPI (c). The composite images are shown in (d), (e), (f) and (g). FEBS Journal 2 (2013) ª 2013 FEBS 4547

10 N-terminus of SRMS and catalytic activity R. K. Goel et al. staining was also observed (Fig. 5B, b,e). Overall, the merged images did not reveal significant co-localization (Fig. 5B, f,g). A similar observation was made with HEK 293 cells (Fig. S3). Furthermore, via immunoprecipitation analysis using anti- sera, endogenous SRMS failed to co-precipitate with endogenous from the MDA-MB-231 and HBL-100 cell lysates, indicating the absence of binding interactions between the endogenous proteins (data not shown). SRMS directly interacts with via its SH3 and SH2 domains SRMS has SH3 and SH2 domains, which, in Src-family kinases, are known to interact with proline-rich motifs and phosphorylated tyrosine residues, respectively. The C-terminal axis of harbours several proline residues and numerous tyrosine residues that are potential targets for phosphorylation by Src-family kinases (Fig. 6A). Because was identified as a potential target of SRMS [11], we first investigated whether both proteins interact. Accordingly, we transiently transfected GFP- and alone or together in HEK 293 cells and subjected the cell lysates to immunoprecipitation with antibodies against and SRMS, followed by immunoblotting with the same antibodies. As shown in Fig. 6B (lane 7), the co-presence of and GFP- in the same immunocomplex was detected reciprocally in the lysates immunoprecipitated with anti- and anti-srms, respectively. To determine whether interacts with DN-SRMS and to confirm that such an interaction between the exogenous GFP-tagged proteins is not a result of GFP dimerizaton, HEK 293 cells, transiently transfected with either pegfp control vector or co-transfected with pegfp and GFP- or GFP- and DN-SRMS, were subjected to immunoprecipitation with anti- sera and immunoblotted with anti-gfp antibodies. It was found that (a) the interaction between the proteins is not via GFP dimerization (Fig. 6C, lane 4) and (b) interacts with DN-SRMS (Fig. 6C, lane 7). These results suggest that SRMS physically interacts with and that the N-terminal region of SRMS, although being indispensible for sustaining its kinase activity, is expendable for binding interactions of the enzyme with its targets. To determine whether SRMS interacts with via its SH3 and/or SH2 domains, we performed in vitro binding assays using glutathione S-transferase ()-fused SH3 and SH2 domains of SRMS. First, we evaluated the association between endogenous and -SH3 using lysates from untransfected HEK 293 cells. Using antibodies against, we observed interactions between -SH3 and (Fig. 6D, left). However, we did not observe such binding interactions with the -SH2 protein (data not shown). Because the SH2 domain interacts with phosphorylated tyrosine residues, we transiently transfected HEK 293 cells with to determine whether ectopically-expressed SRMS promote the binding of endogenous to the SH2 domain of SRMS. -SH2 was found to form a complex with tyrosine phosphorylated endogenous in the presence of (Fig. 6E,H, left). However, a SRMS -SH2 association with endogenous was not observed in the presence of exogenously expressed pegfp vector or K-M (kinase dead) (Fig. 6F,G) demonstrating that tyrosine phosphorylation is necessary for interactions with SRMS -SH2. The expression and molecular sizes of the and -SH3 and -SH2 proteins are shown by Coomassie Blue staining and immunoblotting with anti- sera (Fig. 6D,E,I). alone bound to beads was used as a control and total cell HEK 293 lysates were used to determine the relative expression of endogenous. Taken together, these data show that both the SH3 and SH2 domains of SRMS mediate interactions with. is a direct substrate of SRMS To characterize and validate as a SRMS substrate, we first transiently transfected HEK 293 cells with GFP-, in the presence or absence GFP- SRMS. was immunoprecipitated from cell lysates using antibodies against and the immunoprecipitates were immunoblotted with antibodies against phosphotyrosine, and SRMS. The results obtained showed an implicit tyrosine phosphorylation of the ectopically-expressed in cells overexpressing SRMS (Fig. 7A, top, lane 4). Autophosphorylation of was also detected in the total cell lysates (Fig. 7A, top, lane 6). In addition, overexpressed SRMS co-immunoprecipitated with as detected by anti-srms serum (Fig. 7A, middle, lane 4), further validating the interaction between and SRMS. The expression of GFP- in the immunoprecipitates is also shown (Fig. 7A, bottom). These data indicate that ectopically-expressed GFP- SRMS interacts with and mediates the phosphorylation of overexpressed GFP-. Upon exogenous expression of in HEK 293 cells, endogenous bound to the purified -SH2 domain of SRMS in a pull-down assay (Fig. 6E). To validate that such interaction was a result of tyrosine phosphorylation of endogenous 4548 FEBS Journal 2 (2013) ª 2013 FEBS

11 R. K. Goel et al. N-terminus of SRMS and catalytic activity A B D E -WT (1-481) : GFP-: IP SRMS Input Input anti- -SH3 anti- PH SH2 PTB Y P2 P282 P283 P292 P293 -SH3 Coomassie P307 P309 Y296 Y315 Y337 Y341 Y362 P370 P372 P373 -SH2 P3 P383 Y377 Coomassie P1 P3 Y398 Y2 Y9 P412 P413 P414 Y449 Blot SRMS SRMS P423 P425 P428 P431 P433 -SH3 -SH2 C F H GFP-ΔN-SRMS: GFP-: pegfp: Input Input IP: pegfp anti- anti-ptyr -SH SH2 G I KM Input anti- -SH2 Blot: GFP GFP- GFP-ΔN-SRMS -SH2 anti- -SH2 Fig. 6. SRMS interacts with in vivo and in vitro. (A) Schematic diagram of depicting its two functional domains, namely the pleckstrin homology (PH) domain and the phosphotyrosine-binding (PTB) domain, as well as a proline and tyrosine-rich C-terminal axis. (B) HEK 293 cell lysates from, GFP- or /GFP- co-transfected cohorts were subjected to immunoprecipitation with anti- and immunoblotted with and SRMS (top). Conversely, SRMS was immunoprecipitated from such lysates using anti- SRMS and the immunoprecipitates probed for SRMS and (bottom). Anti-IgG (rabbit) was used as the control. Total cell lysates indicate the relative expression of the proteins. (C) Cell lysates were subjected to immunoprecipitation with anti- in the GFP-/pEGFP or GFP-/GFP-DN SRMS co-transfected cohorts and immunoblotted with antibodies against GFP to probe for GFP dimerization-mediated interactions between the ectopic proteins. (D) Endogenous from HEK 293 lysates binds to the purified recombinant -SH3 domain of SRMS in a pull-down assay, as demonstrated upon immunoblotting with anti (left). Expression of the bacterially expressed proteins is shown via Coomassie Blue staining (right). (E) Tyrosine phosphorylated endogenous from -transfected HEK 293 cell lysates binds to the -SH2 domain of SRMS, as shown via immunoblotting with anti-. Expression of the -fused proteins is shown in the Coomassie Blue stained image of the gel (right). (F) Tyrosine phosphorylation of endogenous upon exogenous expression of was probed with phosphotyrosine antibodies. (G, H) Endogenous binding to -SH2 domain of SRMS was examined upon exogenous expression of the pegfp vector control or -K258M (kinase inactive mutant). (I) The expression of the -fused proteins is shown via blotting with anti- sera. FEBS Journal 2 (2013) ª 2013 FEBS 4549

12 N-terminus of SRMS and catalytic activity R. K. Goel et al. A GFP- B GFP-ΔN-SRMS : IP: 100 IgG TCL IgG TCL WB: ptyr GFP- IP: IgG anti- GFP-ΔN-SRMS anti-gfp WB: SRMS anti-ptyr IgG WB: DOK1 GFP- IgG C WT (1-481) Dok Δ1(1-259) PH PTB PH Y146 Y146 PTB Y296 Y315 Y337 Y341 Y362 Proline-rich Y377 Y398 Y2 Y9 Y449 Dok Δ2(1-317) PH PTB Dok Δ3(1-345) PH PTB Dok 4(1-3) PH PTB Dok Δ5(1-415) PH PTB D E F mcherry-srms: mcherry-srms Y296 Y315 Y337 Y341 Y362 Y377 Y398 Y9 Y2 GFP-: WB: anti-ptyr anti-gfp (r) anti-srms GFP IP: anti-gfp (r) WB: anti-ptyr anti-gfp (m) anti-srms - : SRMS : ATP : anti-ptyr anti- - -SRMS - -SRMS 45 FEBS Journal 2 (2013) ª 2013 FEBS

13 R. K. Goel et al. N-terminus of SRMS and catalytic activity Fig. 7. is a bonafide substrate of SRMS. (A) was immunoprecipitated from GFP- alone or GFP-/ cotransfected cohorts of HEK 293 cell lysates and probed for tyrosine phosphorylation against phosphotyrosine antibodies (top), SRMS (middle) and (bottom). Anti-IgG (rabbit) was used as a control. Total cell lysates were used to indicate the relative expression of both proteins. (B) was immunoprecipitated from or GFP-DN-SRMS transfected cohorts of HEK 293 cell lysates and immunoblotted for (top), SRMS (middle) and phosphotyrosine (bottom). Total cell lysates were used to indicate the relative expression of the respective proteins. Anti-IgG (rabbit) was used as a control. (C) Schematic representation of the 5 mutants that were constructed from wild-type GFP-(Dok-WT). Each C-terminally truncated mutant contains an increasing number of tyrosine residues. (D) mutants were transfected either alone or with mcherry-srms in HEK 293 cells and total cell lysates were used for immunoblotting with antibodies against phosphotyrosine (top), GFP (middle) and SRMS (bottom). (E) HEK 293 cells were co-transfected with the mutants and mcherry-srms and subjected to immunoprecipitation with anti-gfp (rabbit) antibodies. Immunoprecipitates were probed for tyrosine phosphorylation using antibodies against phosphotyrosine (top), using anti-gfp (mouse) (middle) and SRMS using anti-srms (bottom). (F) An in vitro kinase assay was performed using the active kinase, -SRMS, and the substrate, -, in the presence or absence of ATP. Tyrosine phosphorylation was probed via anti-phosphotyrosine sera (top) and expression of the -fused proteins via anti- (bottom). by, we transiently transfected or DN-SRMS in HEK 293 cells and immunoprecipitated via anti- sera. The immunoprecipitates were then immunoblotted with antibodies against phosphotyrosine, SRMS and. We demonstrate that binds to and phosphorylates endogenous (Fig. 7B, middle, lane 4; bottom, lane 4). Furthermore, the finding that DN- SRMS co-immunoprecipitated with endogenous corroborates the results shown in Fig. 6C, attesting to the dispensability of the 51 amino acid N-terminus sequence with respect to regulating SRMS substrate recognition and interaction. As shown in Fig. 6A, possesses a dense array of prolines and tyrosine residues along its C-terminal segment. In an attempt to map the cluster of tyrosine residues phosphorylated in the presence of SRMS, we generated five deletion mutants of, each containing a progressively increasing number of tyrosine residues (Fig. 7C). Wild-type and its mutants were transiently transfected in HEK 293 cells, either alone or with mcherry-srms. The mutants were immunoprecipitated with anti-gfp sera and the immunoprecipitates and total cell lysates were immunoblotted with antibodies against phosphotyrosine, GFP and SRMS. Immunoblotting the total cell lysates (Fig. 7D, top) revealed that, besides wild-type (lane 12), Dok-D3 (1-345), Dok-D4 (1-3) and Dok- D5 (1-415) were all phosphorylated in the presence of mcherry-srms. Barring Dok-D1(1-259) as the only exception, Dok-D2 (1-317) was found to be phosphorylated upon longer exposure of the X-ray film to the immunoblotted membrane (data not shown). The expression levels of transfected GFP- mutants (Fig. 7D, middle) and mcherry-srms proteins (Fig. 7D, bottom) are shown. Immunoblotting performed on the immunoprecipitates also demonstrated identical results (Fig. 7E), which confirmed that all mutants, except Dok-D1, were phosphorylated in the presence of ectopically-expressed mcherry- SRMS. Furthermore, the finding that co-immunoprecipitation of ectopic SRMS progressively diminished with the smaller mutants (Fig. 7E, bottom, lanes 1 6) highlights the underlying significance of the C-terminal proline residues in mediating binding interactions with SRMS. Taken together, these data provide strong evidence that is a potential substrate of SRMS. To determine whether was a direct substrate of SRMS, we performed an in vitro kinase assay. We incubated -SRMS and - in the presence or absence of ATP in a kinase reaction. The proteins were resolved by SDS/PAGE and analyzed by immunoblotting using anti-phosphotyrosine sera. It was found that - was strongly phosphorylated in the presence of -SRMS and ATP (Fig. 7F, lane 3, top). The activity of SRMS was confirmed by the autophosphorylation of -SRMS in the presence and absence of - (Fig. 7F, lanes 3 and 1). Immunoblotting with anti- (Fig. 7F, bottom) was used as a control for the presence of the -tagged proteins. The data reported in the present study are the first to indicate that is a direct and bona fide substrate of SRMS. Discussion The nonreceptor tyrosine kinase, SRMS, was cloned in 1994 but, subsequently, has remained understudied. Nothing is known or has been reported about its enzymatic activity, substrate identification and recognition, nor its expression profile in human tissues, cell lines or carcinomas. The urgency to study this mysterious protein appeared to be even more compelling because SRMS shares a conservative structural commonality with its other well-characterized family members, BRK and FRK. The present study is the first of its kind to investigate and unravel (a) the expression profile of FEBS Journal 2 (2013) ª 2013 FEBS 4551

14 N-terminus of SRMS and catalytic activity R. K. Goel et al. SRMS in breast cancer cell lines and tissues and (b) the mechanisms for SRMS autocatalytic regulation and substrate specificity. Along these lines, we have established that: (a) SRMS is overexpressed in the breast cancer cell lines studied and breast carcinomas but is found to be low in human mammary epithelial cells obtained from normal tissues and normal mammary tissues; (b) the endogenous and ectopicallyexpressed SRMS localizes to punctate cytoplasmic structures; (c) the N-terminal region of SRMS is an essential element regulating its enzymatic activity; and (d) is a cellular target and a direct substrate of SRMS. SRMS was originally cloned from the murine embryonic neuroepithelial cells and the mouse skin, and was found to be strongly expressed in mouse lung, testes and liver, in addition to the epithelial keratinocytes [6,7]. Our data show that SRMS is expressed or overexpressed in all human breast carcinoma samples tested compared to BRK whose expression is observed in most (but not all) cell lines (Fig. 1). However, we detected only diminutive SRMS expression in the nontumourigenic/normal breast epithelial cell line, 184B5, and in normal tissues. The SRMS gene, similar to that of BRK, comprises eight exons and, because the former localizes to chromosome 20q13.33, mapping within 1.5 kbp upstream of BRK, both PTK genes remain tightly linked [8]. This locus, notably, resides in a region of the genome that is frequently amplified in breast cancers [36]. Studies on BRK have shown that the protein is expressed in more than % of breast tumours and breast cancerderived cell lines, whereas it is absent in normal mammary tissues and benign lesions [8]. BRK is also overexpressed in various other types of cancers, including those of the ovary, colon, head and neck, and prostate [9,37 41]. The dramatic induction of BRK in a significant percentage of human breast tumours suggests a role for BRK in the aetiology of breast cancers. BRK has oncogenic properties and can promote cell proliferation, migration and tumour formation [8,20]. Consistent with its potential role in tumourigenesis, BRK has also been shown to associate with epidermal growth factor receptor, as well as enhance the mitogenic signals of EGF and promote the recruitment of phosphatidylinositol 3-kinase, in addition to activating Akt and mitogen-activated protein kinase [42,43]. By contrast, the other BRK family protein, FRK, is a putative tumour suppressor in breast cancer and has been shown to inhibit cell growth and suppress tumourigenesis [44]. One mechanism of action of FRK is via the phosphorylation and stabilization of the tumour suppressor phosphatase and tensin homologue, which results in the inhibition of Akt signalling [45]. The human FRK gene maps to the chromosomal locus 6q21-23, a region that is destabilized by a loss of heterozygosity in 30% of breast tumours [46] and % of melanomas [47]. Our data from IHC analysis indicate that SRMS expression is associated with the mechanistic onset of human breast cancer and is apparently gradually augmented in vivo as the disease aggravates with time (Fig. 1B). This is also indicative of an intricate involvement of SRMS in the subcellular molecular aberrations occurring during the progressive transformation from a presumably early lesion-type condition to a full-blown cancer phenotype. Although the present study is the first to examine SRMS expression in breast cancer samples, given the significance of the investigation s outcome, it is highly likely that, similar to BRK and FRK, induction of SRMS in breast cancer correlates with the aetiology of the disease. Although SRMS has three functional domains (an SH3 domain, an SH2 domain and a tyrosine kinase catalytic domain) that are conserved in Src family kinases, the primary amino acid sequence of SRMS shows the presence of a 51 amino acid extended N-terminal region. Another striking characteristic of SRMS is the absence of a C-terminal regulatory tail present in most Src-family kinases, including BRK and FRK. In addition, unlike the Src-family members, SRMS lacks myristoylation and palmitoylation membrane-anchoring signals, which potentially renders the protein a reasonable degree of flexibility in subcellular localization, and therefore also bestows upon it a broader access to potential cellular targets. We found that both endogenous and ectopically-expressed SRMS localizes to punctate cytoplasmic structures (Fig. 2A,B), which was demonstrated even with fractionation studies (Fig. 2C). However, because the localization of wildtype SRMS is characteristically punctate as opposed to diffused, we suggest that such localization is tightly regulated and that the protein anchors to its location via interactions with specific endogenous protein targets. Our results show that the localization of SRMS may be dictated by interactions involving the extended N-terminus or the SH2 domain (Fig. 4). The deletion of any of these regions at least partially altered the cytoplasmic localization of SRMS from punctate to diffuse. It is possible that the N-terminal region and the SH2 domain mediate intermolecular interactions with other target proteins that sequesters SRMS to distinct punctuate structures within the cell. However, how these critical regions mediate such associations with other proteins is as yet unknown. Intermolecular interactions involving the SH2 and SH3 domains of 4552 FEBS Journal 2 (2013) ª 2013 FEBS