Application of IMAC to the analysis of RhoE phosphorylation

Several experiments performed by Kirsi Riento (Cellular and Molecular Biology, LICR) indicated that phosphorylation of RhoE by ROCK can take place (Riento, 2003). Firstly, co-expression of RhoE and ROCK in Cos? cells induced a shift in the

Chapter 4: Analysis of Signal Transduction Proteins

electrophoretic mobility of RhoE, and secondly, in vitro ROCK kinase assay using recombinant RhoE as a substrate also indicates that RhoE is a substrate for ROCK (K Riento, Personal Communication). Further experiments indicated that phosphorylation only occurred if the N-terminal portion of the protein was present within expression constructs, and that phosphorylation was further reduced by C- terminal truncation o f the protein (K Riento, Personal Communication). The analysis of RhoE using IMAC and mass spectrometry was initiated as a final year undergraduate project carried out by Hanako Tsushima (UCL Department of Biochemistry). In the initial study carried out by K. Riento and H. Tsushima, Flag- tagged RhoE constructs were co-transfected into Cos7 cells with a fusion vector encoding a myc-tagged version of the ROCK kinase. Protein was immunoprecipitated and separated by ID gel electrophoresis and peptide mass mapping o f in-gel digests was carried out to confirm the identity o f the observed 27.5 kDa band as RhoE. Initial IMAC separation and MALDI-MS analyses did not conclusively indicate phosphorylation of the protein. I therefore continued the initial collaborative study, applying alternative methods as we attempted to discover the phosphorylation sites using a gel-ffee approach.

The gel-free approach for preparing RhoE samples was performed as described in Chapter 2. Briefly, the RhoE was overexpressed as a GST fusion protein in E.coli, as previously described (Guasch, 1998c; Riento, 2003). This allowed affinity purification of RhoE upon glutathione-sepharose beads without the use of antibodies, which are known to cause difficulties with mass spectrometric analyses due to the presence of high levels o f contaminant, antibody-derived peaks in samples where electrophoretic separation is not used. Overexpressed GST-RhoE, (purified using GSH sepharose and eluted by thrombin cleavage of the fusion tag), was phosphorylated by co-incubation with immunoprecipitated, myc-tagged ROCK, before centrifugation to pellet the beads and removal of supernatant. This supernatant, as provided by K. Riento, was digested and subjected to peptide mass mapping and IMAC treatment. The sequence of RhoE had indicated that the use of trypsin was inappropriate in this study, as clusters of lysine and arginine residues were present within the protein N- and C-termini, where predicted phosphorylation sites were thought to be present. However, Asp-N digestion yielded a very large peptide (theoretical m/z 4794.69 for the peptide Aspi8i-Thr2 3i) in the C-terminal portion o f the

protein, which was outside of the range that is readily resolved by MALDI and ESI- MS. Endoproteinase Lys-C was therefore used as an intermediate between these two extremes.

MALDI analysis of the RhoE protein before and after IMAC treatment as presented in Figure 4.3.2, indicated four putative phosphorylation sites, two residues within an N- terminal peptide, one singly- and one doubly-phosphorylated peptide at the C- terminus o f the protein (as indicated in Figure 4.3.2 and Table 4,3.1). MALDI-TOF- PSD analysis could only give extremely limited information about the C-terminal phosphorylation site within peptide ion P2, and insufficient signal was gained from other peaks to be able to carry out PSD experiments. On-target CIP treatment yielded no further information regarding these samples, as the increased salt concentration following CIP treatment meant that no peptide signals could be seen by MALDI. Attempts to carry out LC-MS/MS upon IMAC-retained RhoE fractions yielded no further information about the phosphorylation sites. Another observation from these experiments was that an unusually high rate of loss o f the postulated phosphopeptides occurs during freeze-thaw cycles, this may have been due to non-specific adsorption of the hydrophilic peptides to the walls of the tubes. Subsequent analyses of the same Lys-C digests which had been subjected to freeze-thawing yielded increasingly poor results, with sequence coverage and putative phosphopeptide signal intensity falling dramatically (data not shown).

The peptides isolated using IMAC as shown in Figure 4.3.2 were all tentatively assigned to serine/threonine-containing peptides which were also within the N- terminal and C-terminal regions o f the protein which previous studies by K. Riento had shown to be required for efficient phosphorylation of RhoE in the presence of ROCK. Although these analyses were capable of giving an initial insight into the possible phosphorylation sites following ROCK phosphorylation o f RhoE, further analysis by site-directed mutagenesis is currently underway to determine if these putative sites are important for phosphorylation or functional behaviour of this protein (K Riento, Personal Communication). Further mass spectrometric analysis using larger amounts o f protein could resolve the phosphorylation sites within this protein. This study illustrated that IMAC/MALDI analysis for the elucidation of

Chapter 4: Analysis of Signal Transduction Proteins

phosphorylation sites should be used as part o f a multi-faceted, targeted approach to the analysis o f protein phosphorylation sites upon specific proteins o f interest.

1.1 E+4 70= CO c 6(^ c 30l 20 01— 727.0 1 406.8 2 0 86.6 2766.4 3446.2 —iO 4126.0 m/z lOOi 0--- 808 1463 2118 2773 3428 —fO4083 m/z

c

P,-98

Figure 4.3.2: Mass spectrometric analysis of RhoE Lys-C digests.

Peptide mass mapping was carried out upon the Lys-C digest shown in A), searching o f the NCBI database gave 23% sequence coverage for RhoE. IMAC separation o f the digest (B) indicated several possible sites o f phosphorylation, two within an N-terminal peptide, sequence ERRASQKLSSK, and two phosphorylated C-terminal peptides RISHMPSRPELSAVATDLRK, and DKAKSCTVM (see Table 4.4.1). PSD analysis o f the peptide P? showed a minor product ion at a mass 98 Da lower than the parent (C).

Table 4.3.1: Putative phosphopeptide ions observed in peptide mass mapping and IMAC experiments as in Figure 4.3.1

Peptide T h eoretical m ass

R esid u es Pu tative S eq u en ce M o d ifi­

cation s S ig n a l pre- /p o st- IM A C C onfirm ation o f p h osp h orylation b y P S D Pi 1 2 2 9 .4 3 4 9 2 4 1 -2 4 9 D K A K S C T V M (-) 2PO4, 1M „, B o th _N N , 2 2 6 4 .2 2 9 7 2 2 1 -2 4 0 R IS H M P S R P E L S A V A T D L R K Pre N /A ? 2 2 3 4 4 .1 9 6 1 2 2 1 -2 4 0 R IS H M P S R P E L S A V A T D L R K I P 0 4 B oth Y P3 1 3 6 9 .6 9 5 3 8 -1 8 E R R A S Q K L S S K I P 0 4 P o st N P4 1 4 4 9 .6 6 1 6 8 -1 8 E R R A S Q K L S S K 2 P 0 4 P o st N N2 3 4 3 2 .6 8 1 8 1 4 2 -1 7 2 S D L R T D V S T L V E L S N H R Q T P V S Y D Q G A N M A Pre N /A