**2.1.3 Phosphorylation on Tyrosine 315 by BCR/Abl**

Rad51 can also be phosphorylated by the oncogenic fusion tyrosine kinase BCR/Abl. BCR/Abl is expressed in most cases of chronic myeloid leukemia and in some cases of acute myeloid leukemia and possesses constitutive kinase activity.

Slupianek and colleagues suggested that Rad51 and BCR/Abl interact physically since a portion of Rad51 co-localizes with the fusion tyrosine kinase in the cytoplasm of BCR/Abl overexpressing cells. This interaction was confirmed by the co-immunoprecipitation of the two proteins.

Rad51 was immunoprecipitated from cells overexpressing BCR/Abl and its phosphorylation state was examined with an anti-phosphoTyrosine antibody. The interaction between

Posttranslational Modifications of Rad51 Protein and Its Direct Partners:

Role and Effect on Homologous Recombination – Mediated DNA Repair 147

Another possible explanation could be the proximity of the digestion and the phosphorylation sites. The presence of phosphorylation near a digestion site may decrease its digestion efficiency (Benore-Parsons et al., 1989; Kjeldsen et al., 2007). Thus the phosphorylated protein would be partially digested resulting in a longer phospho-peptide. A corresponding peptide would not be obtained from the digestion of the unphosphorylated protein. A phosphorylation peak would not be observed in these conditions. In the amino acid sequence of Rad51, only one residue separates the trypsin digestion site from Tyrosine 315. Due to the proximity of the two sites, Rad51 would rather be digested at arginine 310 than on lysine 313. This would result in the generation of a phosphopeptide which would be 3 amino acids longer than the corresponding peptide from the unphosphorylated protein. Consequently, the

Popova and co-authors have established a possible mechanism by which Rad51 is phosphorylated by c-Abl. They co-expressed c-Abl and wild type or mutated hRad51 in cells. In the amino acid sequence of hRad51, Tyrosine 54 or Tyrosine 315 were mutated to phenylalanine, thus rendering the residue at this position nonphosphorylatable. Western blot analysis of the cell lysates, revealed with their specific anti-phosphoTyrosine antibodies, showed a relationship between the phosphorylation of Y54 and Y315. When residue 315 was mutated to phenylalanine and nonphosphorylatable, Tyrosine 54 was no longer phosphorylated. On the contrary, the mutation of residue 54 had no effect on the phosphorylation of Tyrosine 315. The authors hypothesized that the phosphorylation of

The c-Abl kinase possesses a SH3 and a SH2 domain in its N-terminal region. The SH3 domain recognizes and binds preferentially to proline rich regions containing the sequence PXXP. The SH2 domain recognizes pYXXP sequences. hRad51 has two PXXP motifs in its amino acid sequence – between amino acids 283 and 286, and between amino acids 318 and 321. When Tyrosine 315 is phosphorylated, a pYXXP motif is revealed between amino acids

According to this model of sequential phosphorylation, c-Abl recognizes a PXXP motif in the sequence of Rad51 through its SH3 domain and phosphorylates Tyrosine 315. The phosphorylation of this residue reveals the pYXXP binding motif which is recognized by the

To confirm this model, GST pull-down assays were performed. A GST- c-Abl SH2 domain peptide was incubated with lysates from cells overexpressing Rad51 and c-Abl. The results showed that hRad51 binds to the SH2 domain of c-Abl and that this interaction takes place when Rad51 is phosphorylated on Tyrosine 315. Therefore a model of sequential phosphorylation of Rad51, where the phosphorylation of Tyrosine 315 by c-Abl reveals a novel binding site for the kinase thus allowing the phosphorylation of Tyrosine 54, is highly

Even though the process of phosphorylation seems to be of considerable importance in the regulation of Rad51 activity, its exact roles and consequences have not been elucidated yet.

In their study, Yuan and colleagues investigated the possible effect of Y54 phosphorylation on Rad51 activity. Strand exchange assays showed that phosphorylation of S. cerevisiae

phosphorylation of Rad51 on Y315 would not be detected by mass spectroscopy.

**2.1.6 Model of sequential phosphorylation** 

plausible.

**2.2 Role of Rad51 phosphorylation** 

Moreover, the existing data is contradictory.

Tyrosine 315 is needed for the phosphorylation of Tyrosine 54.

315 and 318. This motif might be recognized by the SH2 domain of c-Abl.

SH2 domain of c-Abl. This allows the phosphorylation of Tyrosine 54.

BCR/Abl and Rad51 resulted in the constitutive phosphorylation of Rad51 on tyrosine. Rad51 was also phosphorylated by c-Abl after treatment of cells with cisplatin and mitomycin C. In order to determine the position of phosphorylation, the authors transiently co-expressed BCR/Abl and wild type or mutated Rad51 in cells. Tyrosine to phenylalanine mutations were performed at Tyrosine 54 or Tyrosine 315. The analysis of the Rad51 immunoprecipitates with an anti-phosphoTyrosine antibody revealed the phosphorylation of the wild type and the Y54F Rad51 protein. A substantial reduction in the phosphorylation level of Rad51 was observed when Y315 was mutated to phenylalanine, indicating that the majority of the phosphorylation of Rad51 occurred on Y315. To further confirm the phosphorylation of the Y315 residue, Slupianek and colleagues prepared an antiserum using a phosphorylated Y315 peptide. Western blots were then performed with lysates from cells overexpressiong Rad51 alone or with BCR/Abl. The antiserum did not recognize Rad51 when the protein was overexpressed in cells alone. In contrast, in cells co-expressing BCR/Abl a strong signal was observed. This confirms that the fusion tyrosine kinase BCR/Abl phosphorylates Rad51 on Tyrosine 315 (Slupianek et al., 2001).

#### **2.1.4 Phosphorylation by Arg**

The only other member of the c-Abl family, the kinase Arg, also phosphorylates Rad51. Arg shares considerable structural and sequence homology with c-Abl in the N-terminal SH3 and SH2 domains, as well as in the tyrosine kinase domain (Kruh et al., 1990). Coimmunoprecipitation of Rad51 from cells overexpressing Rad51 and Arg indicated that Arg can interact with Rad51 *in vivo*. An anti-phosphoTyrosine antibody showed that Rad51 is phosphorylated by Arg and this phosphorylation seemed to be more effective than the phosphorylation by c-Abl. However, the position of phosphorylation was not determined (Li et al., 2002).

#### **2.1.5 Phosphorylation of both Tyrosine 54 and Tyrosine 315 by c-Abl**

The study conducted by Popova and colleagues has allowed to reconcile the discrepancies on which tyrosine residue is phosphorylated in Rad51. The authors purified specific antiphosphoTyrosine antibodies for each site of phosphorylation. These antibodies were used to analyze the phosphorylation state of Rad51 by immunoblotting of lysates from cells overexpressing Rad51 and c-Abl. The ability of these specific antibodies to detect distinctively the phosphorylation of the two tyrosine residues has allowed to observe the phosphorylation of both Y54 and Y315 in the same experiment. This confirmed that both Tyrosine 54 and 315 can be phosphorylated (Popova et al., 2009).

In all previous studies the phosphorylation of only one site was observed, either Y54 or Y315. The fact that Yuan and colleagues observed only the phosphorylation of Y54 and did not detect the phosphorylation of Y315 could be due to the technique they used. In their study, the *in vitro* or *in vivo* phosphorylated Rad51 protein, as well as the unphosphorylated protein were digested by trypsin. The obtained fragments were then analyzed by mass spectroscopy and the spectra of the unphosphorylated and the phosphorylated proteins were compared. The lack of a phosphorylation peak in the fragment containing Y315 could be explained by its biophysical characteristics. Following trypsin digestion, the peptide containing Tyrosine 54 is 17 amino acids long and has a pHi of 4,83. On the contrary, the peptide containing Tyrosine 315 is 28 amino acids long and its pHi is 4,03. Thus, the Y315 peptide is longer and more negatively charged compared to the Y54 peptide which could interfere with its detection by mass spectroscopy (Raggiaschi et al., 2005).

Another possible explanation could be the proximity of the digestion and the phosphorylation sites. The presence of phosphorylation near a digestion site may decrease its digestion efficiency (Benore-Parsons et al., 1989; Kjeldsen et al., 2007). Thus the phosphorylated protein would be partially digested resulting in a longer phospho-peptide. A corresponding peptide would not be obtained from the digestion of the unphosphorylated protein. A phosphorylation peak would not be observed in these conditions. In the amino acid sequence of Rad51, only one residue separates the trypsin digestion site from Tyrosine 315. Due to the proximity of the two sites, Rad51 would rather be digested at arginine 310 than on lysine 313. This would result in the generation of a phosphopeptide which would be 3 amino acids longer than the corresponding peptide from the unphosphorylated protein. Consequently, the phosphorylation of Rad51 on Y315 would not be detected by mass spectroscopy.
