1 Department of Cell Biology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA
2 Department of Chemistry, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA
3 Department of Pathology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA
4 Department of Biology, Centenary College of Louisiana, 2911 Centenary Avenue, Shreveport, LA 71104, USA
Author for correspondence (e-mail: donna.webb{at}vanderbilt.edu)
Paxillin is a 68 kDa protein that was originally identified as a substrate for the non-receptor tyrosine kinase Src (Glenney and Zokas, 1989; Turner et al., 1990
). Paxillin localizes primarily to specialized sites of adhesion between cells and the extracellular matrix and functions as an adaptor molecule that recruits signaling and structural proteins to these sites through its multiple domains (Brown and Turner, 2004
; Turner et al., 1990
; Turner, 2000a
). The N-terminal half of paxillin contains five short, leucine-rich peptide sequences (LDXLLXXL), which mediate interactions with other proteins (Brown et al., 1998a
), and a proline-rich region that provides a binding site for the Src homology 3 (SH3) domains of Src family members (Weng et al., 1993
). Phosphorylation of several tyrosine residues in this region creates binding sites for proteins containing SH2 domains (Bellis et al., 1995
; Schaller and Parsons, 1995
; Turner and Miller, 1994
). The C-terminal half of paxillin contains four LIM domains that also facilitate protein-protein interactions (Turner and Miller, 1994
). Phosphorylation of serine and threonine residues in this region potentiates the localization of paxillin to adhesions (Brown et al., 1998b
). Thus, it appears that the localization of paxillin and its function as an adaptor molecule are both regulated by phosphorylation.
In this study, we used mass spectrometry to map tyrosine, serine and threonine phosphorylation sites in paxillin. Samples were prepared by transfecting HEK cells with either FLAG- or FLAG-GFP-tagged paxillin (10 ng to 3.5 µg per 100 mm dish) followed by immunoprecipitation of these FLAG-tagged molecules with FLAG-agarose (Sigma). Before cells were lysed, they were treated for 30 minutes with peroxovanadate (1 mM) and calyculin A (10 nM), a tyrosine and a serine/threonine phosphatase inhibitor, respectively. In the absence of phosphatase inhibitors, several peptides containing phosphoserine and phosphothreonine residues were observed at reduced levels. Under the same conditions, peptides phosphorylated on tyrosine often went undetected (Table 1).
|
Immunoprecipitated FLAG-tagged paxillin was digested with either tryspin, chymotrypsin, trypsin/chymotrypsin or Glu-C in an effort to generate peptides that provided complete coverage of the protein sequence. These peptides were analyzed by using high performance liquid chromatography (HPLC) interfaced to electrospray ionization on tandem mass spectrometers (LCQ-XP or LTQ-FT, Thermo Electron). Enrichment of phosphopeptides was performed using immobilized metal affinity chromatography (IMAC) (Ficarro et al., 2002). With these techniques, we obtained greater than 97% coverage of the serine, threonine and tyrosine residues in paxillin (Fig. 1). Ten of the phosphorylation sites that we identified had been described previously (tyrosines 31, 40, 88, 118 and 182, serines 85, 126, 130 and 188/190, and threonine 403) (Bellis et al., 1995
; Bellis et al., 1997
; Brown et al., 1998b
; Huang et al., 2004
; Schaller and Parsons, 1995
; Schaller and Schaefer, 2001
; Turner and Miller, 1994
; Turner, 2000b
; Woodrow et al., 2003
). Phosphorylation of serine 85 and tyrosines 31 and 118 regulates cell migration (Huang et al., 2004
; Petit et al., 2000
). The functional significance of the other sites remains to be determined.
|
Several of the novel phosphorylation sites are potential targets for kinases that are implicated in the regulation of various cellular processes including migration and adhesion. Serines 91, 98, 108, and 382 and threonines 295 and 540 are predicted sites for protein kinase C (PKC), while serines 112, 173, 217, 259 and 501 are predicted substrates for protein kinase A (PKA) (Table 1). GSK3 is a predicted kinase for serines 85, 90, 94, 106, 108, 126 and 227, and serine 173 is a predicted site for Akt. Although earlier studies focused on the function of Akt in apoptosis, emerging evidence suggests that this protein kinase also plays a key role in cell migration (Brazil et al., 2002). Extracellular signal-regulated kinase (ERK), which has recently been shown to localize to adhesions and to regulate paxillin disassembly (Fincham et al., 2000
; Webb et al., 2004
), is predicted to phosphorylate several sites in paxillin (S106, S231 and S290). Whether these sites also play a role in the regulation of cell migration and adhesion dynamics remains to be determined. Many residues that are phosphorylated in chicken paxillin are conserved in human and mouse paxillin (hpax and mpax) and in another paxillin family member (hic-5) (Table 1).
A selection of paxillin peptides that contain more than one phosphorylated residue are shown in Table 2. All nine of these peptides contain phosphorylation sites separated by 0-8 residues. In several of these peptides, both the phosphorylation sites and the intervening residues are conserved in chicken, mouse and human paxillin. We suggest that these sequences function as multiply phosphorylated, regulatory or recognition motifs. As shown in Table 2, two peptides contain adjacent phosphorylated tyrosine and serine residues (Y118/S119 and Y88/S89). Although the simultaneous phosphorylation of adjacent tyrosine and serine residues has not been previously described, it seems likely that this combination serves a regulatory function within the molecule. Phosphorylation of an adjacent serine residue could block protein-protein interactions that recognize the phosphotyrosine site and therefore act as an on-off switch for the formation of such complexes. A similar phenomenon is known to exist in histones, where proteins that recognize methylated lysine residues are blocked by phosphorylation of adjacent serine or threonine residues (Fischle et al., 2003).
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Of additional interest is the observation that 50% of the paxillin molecules are modified with an O-linked N-acetylglucosamine (GlcNAc) moiety on serine 74. This type of modification is reversible and thought to have a modulatory function similar to that of phosphorylation (Hart, 1997). GlcNAc modifications on proteins can occur at the same sites as those that are phosphorylated, but the modification is not restricted to these residues (Comer and Hart, 2000
). In the present study, we did not detect phosphorylation of serine 74.
Although the phosphorylation map presented here is extensive, it may not be complete. For example, we detected phosphorylation on all three of the predicted ERK sites, but not on two potential PKC sites (S19 and T511) and one PKA site (T516). This raises the possibility that other phosphorylation sites exist. Additional phosphorylation sites could be generated under different growth conditions or with other cell types. It is also possible that some sites are phosphorylated at very low levels, owing to high spatial and temporal regulation. In this context, some sites were only observed in a single experiment (T29 and S143). Finally, although we have not assigned relative abundances of the phosphopeptides detected in the present work, those detected without inhibitors or IMAC are among the most abundant. The least abundant phosphopeptides are those that are only detected in the presence of phosphatase inhibitors and require enrichment via IMAC prior to analysis by mass spectrometry.
Materials and Methods
Sample preparation
HEK cells were transfected with FLAG-tagged paxillin or FLAG-GFP-paxillin (10 ng to 3.5 µg per 100 mm dish) using lipofectamine. After 36-48 hours, cells were incubated with 1 mM peroxovanadate and 10 nM calyculin A for 30 minutes and extracted with 25 mM Tris, 100 mM NaCl, 0.5% NP-40, pH 7.4. The lysates were precleared twice with mouse IgG-agarose for 1 hour at 4°C and immunoprecipitated with FLAG-agarose (Sigma) for 2 hours at 4°C. Samples were washed twice with 25 mM Tris, 100 mM NaCl, pH 7.4 and FLAG-tagged paxillin was eluted by incubation of the beads with 0.2 mg/ml FLAG peptide in 25 mM Tris for 30 minutes at 4°C or left on beads.
Sample analysis
Mapping of the phosphopeptide sites was performed as described in detail elsewhere (Schroeder et al., 2005). Briefly, FLAG-eluted samples were reduced and alkylated with dithiothreitol and iodoacetamide, respectively, as described previously (Schroeder et al., 2004
). Generally, 10% of the eluted protein was digested with 500 ng of desired enzyme(s) in 100 mM ammonium bicarbonate, pH 8.5 for 8-12 hours at room temperature. Peptides from an aliquot corresponding to 10% of the solution digest (1% of the original IP) were separated with either a 1 or 2 hour gradient as described elsewhere (Schroeder et al., 2004
). Analysis of FLAG-eluted samples was carried out using an LCQ-XP under conventional MS/MS mode (Schroeder et al., 2004
). Reduction and alkylation steps were omitted for on-bead digestion (500 ng trypsin only, shaking at room temperature for 6 hours) and analysis was with an LTQ-FTMS. The on-bead digestion protocol did not reduce peptide coverage, but an intra-peptide disulfide bond was common among peptides containing two cysteines. Enrichment of phosphopeptides was performed according to (Ficarro et al., 2002
) using 10-20x more sample except 250 mM ascorbic acid was used for phosphopeptide elution. ETD spectra were recorded on an in-lab modified LTQ mass spectrometer described in (Syka et al., 2004
) with fluoranthene as the electron transfer reagent.
Acknowledgments
This work was supported by NIH The Cell Migration Consortium (U54 GM064346) and grant GM37537 (D.F.H.).
Footnotes
* Present address: Department of Biological Sciences and Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37235, USA
These authors contributed equally to this work
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