Selección de una celda Peltier

A lthough no peptide sequence homologous to WASP w as obtained from the larger p65 band of the doublet (Table 5.1), the peptide elution profiles for both p63 an d p65 following Lys. C digestion w ere very similar (N. Totty, personal communication), im plying that they are variants of the same protein. For this reason further sequencing of p65 was not perform ed.

CHAPTER 5 CHARACTERISATION OF p63/p65 1 M S G G P M G G R P G G R G A P A V Q Q N I P S T L L Q D H E N Q R L F E M L G R K C L T L A T A V V Q L Y L A L P P G F44 61 A E H W T B E H C G A V C F V F D N P Q K S Y F I R L Y G L Q A G R L L W E Q E L Y S Q L V Y S T P T P F F H T F A G D F71 121 D C Q A G L N F A D E D E A Q A F R A L V Q E K I Q K R N Q R Q S G D R R Q L P PP P T P A N E E R R G G L P P L P L H F38. 181 P G G D Q G G P P V G P L S L G L A T V D I Q N P D I T S S R Y R G L P A P G P S P A D K K R S G K KKISKAjDIGA ..F38(cont) F76 241 P S G F K HVSHV G W D P C N G F D V N N L D P D L R S L F S R A G I S E A Q L T D A E T S K L I Y D F I E D Q G G L 3 01 E A V R Q E M R R Q E P L P P P P P P S R G G N Q L P R P P I V G G N K G R S G P L P P V P L G I A P P P P T P R G P P 361 P P G R G G P P P P P P P A T G R S G P L P P P P P G A G G P P M P P P P P P P P P P P S S G N G P A P P P L P P A L V 421 P A G G P G P G G G R G A L L D Q I R Q G I Q L N K T P G A P E S S A L Q P P P Q S S E G L V G A L M H V M Q K R S R A 481 I H S S D E G E D Q A G D E D E D D E W DD

Fig. 5.2 Amino acid sequence of WASP. Shaded boxes indicate hom ology to peptides generated from m icrosequencing the p63, p65 doublet.

CHAPTER 5__________________________________________ CHARACTERISATION OF p 6 3 /p 6 5

Furtherm ore, peptide sequence corresponding to the F76 peptide derived from the sm aller p63 protein precipitated by the Btk SH3 dom ain (Table 5.1) was present in peptide sequence derived from the heavier p65 species bound by the Fgr SH3 dom ain (Banin et al., 1996), show ing that both proteins of the doublet shared WASP sequences. The nature of the difference betw een the tw o proteins of the doublet is discussed further in section 5.4.

5.2.3 Features of W ASP

WASP is the protein m utated in WAS (section 1.10), w hich like XL A is an X- linked imm unodeficiency disorder. The finding that the Btk SH3 dom ain bound WASP w as therefore of great interest as it indicated a potential link betw een proteins involved in crucial haem atopoietic cell functions.

WASP has a predicted molecular w eight of 54 kDa, w hich is significantly less than that estim ated for the doublet (Fig. 5.1). WASP does not contain any glycosylation sites and so extensive glycosylation w ould not explain this discrepancy. Flowever, WASP contains a large proportion (18%) of proline residues which are know n to be able to retard the mobility of proteins on SDS-PAGE. Such an effect is believed to account for the difference in the predicted size of c-Cbl (100 kDa) and that observed on SDS-PAGE (120 kDa) (Blake et al., 1993).

The proline residues in WASP are arranged in two clusters (section 1.10.2) w hich contain m any putative SH3 dom ain binding sites. The contribution of proline rich sequences tow ards the Btk SH 3/W ASP interaction is discussed in section 5.7. Like c-Cbl, WASP contains no recognisable catalytic activity b u t unlike c-Cbl has no know n transform ing potential.

CHAPTER 5__________ CHARACTERISATION OF p 6 3 /p 6 5

5.3

THE p63, p65 DOUBLET CONTAINS A PROTEIN