CATEGORÍA 2 ANA TERESA ARANDA COMO CANDIDATA INDEPENDIENTE AL GOBIERNO DEL ESTADO DE

(su b d om ain ) PKA R esidue C orresp on d in g residue in PtdlnsPK l i a

P utative function R eferen ce P -lo o p G 5 0 P hosp hate anchor: K n ig h to n et

(I) '151 G 5 5 bin ds to (3- al., (1 9 9 1 )

G 5 2 A I 2 8 p h osp hate o f A T P

S53 R I 2 9

F 5 4 S 130

G 55 G I 3 I

R 5 6 A 132

ATP binding site A 'I'P /F S B A bin d in g Z o lle r et

(II) A 7 0 1143 site: al., (1 9 8 1 )

M 7I 1144 K 7 2 ion pairs w ith a - K n ig h to n et

K 7 2 K l 4 5 and (3-ph osphates and a /., (1 9 9 1 )

fo rm s salt b rid ges w ith Z h e n g et

D I 6 6 and D I 8 4 ai, (1 9 9 3 )

C atalytic loop RI 6 5 R |6 5 ion pairs w ith K n ig h to n et

(V Ib) D 166 D 2 I 2 _{( P ) I 9 7 . 1)1 6 6} a /., (1 9 9 1 )

L I6 7 L 2 I3 _{proposed catalytic} Z h e n g et

K I 6 8 K 2 I 4 _base. a/., (1993)

PI 6 9 _{K 168 ion pairs w ith}

H 170 _{Y -p hosp hate.}

N 171 _{N I 7 I H -b on d s to}

N 166 and a lso lig a tes to IVlg2+ in the a c tiv e site .

M g^ + binding 1 )1 8 4 1 )2 7 3 1 )1 8 4 c h e la te s Mg^+ K n igh ton et

lo o p F 185 Y 2 7 4 al.,{\99\) (V II) G 186 S275 Z h en g et a /.. (1 9 9 3 ) glycine rich ATP catalytic

loop Mg2 4- peptide binding

/catalysis

Myr- II VIb VII 3 5 0

ATP binding c a t a l y s i s

conserved catalytic core

Table 4.1 Com parison of the function of PKA and P td ln sP K I l a resid u es

R esid u es in b old type are h ig h ly conserved a m o n g st m em b ers o f the protein and Pi k in a se su p erfam ily and m any other p h osp hotransferases, those in red are conserved betw een PK A and the P td ln sP -k in a ses. S h o w n b elo w is a sch em atic representation o f the prim ary structure o f P K A , illu stra tin g the subdom ain organ isation .

resulting in the modification of tyrosine, cysteine and histidine residues (Bullough and Allison, 1986; Esch and Allison, 1979; Likos and Colman, 1981; Zoller and Taylor, 1979) and perhaps serine residues (Colman, 1983). Competition with MgATP has been used to demonstrate the labelling specificity of nucleotide analogues (Zoller and Taylor, 1979), however complete protection with ATP was not observed in this study (data not shown) suggesting that enzymatic activity can be destroyed by modification of other sites in the kinase core. Examination of the PtdlnsPK I la sequence indicates the presence of several highly conserved lysine and tyrosine residues which are potential targets for FSBA and could account for this phenomenom. Finally, the predicted size of the (CBS)- peptide lies outside the optimum range of the Lasermat instrument.

4.2.3 Site-directed mutagenesis

In order to demonstrate the functional importance of K145, the putative FSBA and nucleotide-binding residue, PCR mutagenesis (Section 2.2.2.e) was used to construct a mutagenic cassette containing a K145A point mutation. After digestion with the appropriate restriction enzymes, these PCR products were ligated into wild type PtdlnsPK I la in pUC-18 and sequenced on both strands. Clones which were free of secondary (PCR-induced) mutations were subcloned into pGFX-KG (Guan and Dixon,

1991) for bacterial expression as GST fusion proteins (Figure 4.6a). Fusion proteins were eluted as described in Section 2.3.2.1 and assayed for PtdlnsPK activity (Figure 4.6b). Mutation of K145 resulted in a complete loss of activity, indicating that this residue was essential for catalysis. This result supported the proposition that K145 represents a nucleotide-binding residue.

K145 was aligned with subdomain II of PKA and, by using the subdomain organisation of PKA, it was possible to map conserved regions of PtdlnsPKs onto similar regions of local homology in PKA corresponding to subdomains I, II, VIb and VII as shown in Figure 4.5. It was proposed that if this model was accurate, mutation of other conserved residues in these putative subdomains would abolish activity. To this end, K91 (a conserved lysine residue lying outside the proposed PtdlnsPK catalytic core) and K213 (a conserved lysine residue predicted to be in subdomain VIb) were mutated to alanine residues as above. It was found that K91A produced a protein with reduced activity (approximately 80% of the wild type), whereas the K213A mutation resulted in a complete abrogation of activity (Figure 4.6b).

4.2.4 Potential SH3-bindlng sites

Two, short, proline-rich sequences were identified in the divergent insert region of PtdlnsPK I la (amino acids 313-328 in Figure 3.1) which had the potential to act as ligands for SH3 domains. As discussed in Section 1.3.3, SH3 domains are found in a large number of PI signalling molecules including PLCy and heterodimeric PI 3-kinases. PtdlnsPK I la is a predominantly cytosolic enzyme which must, presumably, be

A 117 kDa y 97 - 67 - 45 - 30 - 7 9 kDa

B

K91A K145A K213A