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Experimental strategies adopted to identify protein ligands for PH domains included the affinity screening of rat brain and liver tissue lysates with GST-PH domain fusions immobilised on glutathione Sepharose and the analyses of direct PH domain-protein interactions with isoforms of PKC and the Py subunits of heterotrimeric G-proteins.

5.2.1.1 Protein affinity chromatography

To screen for protein ligands from selected tissue and cell extracts, GST-fusion PH domains from dynamin, Sos, Btk, rasGAP and IR Sl were coupled to glutathione sepharose beads, which were then washed to remove unbound fusion protein. An

important consideration for any such affinity chromatography experiment is the purity of the fusion protein, otherwise any interacting protein that is detected might be binding to a contaminant from the fusion protein preparation. Thus subsequent to coupling of the PH domain fusion to the glutathione sepharose beads, the affinity matrix was washed extensively in a 1% Triton X-100 buffer to remove as much non-specifically bound protein as possible. The matrix was then used to screen 1% Triton X-100 solubilised extracts of rat brain and liver tissues, human monoblastic leukaemic cell line U937 and HELA cells for putative PH domain-binding proteins. The matrix coupled PH domains recovered from the lysates were washed, and complexed proteins resolved by SDS- PAGE were visualised by silver staining. GST-Grb2 was used as a positive control in the affinity screening of rat brain lysates, since it had previously been shown to affinity purify dynamin from the aforementioned extract (Gout et al., 1993). The two cell lines were selected for screening on the basis that they were a readily available source within the laboratory and could be routinely cultured. This protein affinity chromatography approach however, did not yield any specific PH domain binding proteins (data not shown).

5.2.1.2 Protein kinase C

The constitutive association of Btk PH domain with both Ca^+-dependent (a, pi and pH) and Ca^+-independent (y and 0 isoforms of protein kinase C (PKC) (Yao et al., 1994) led to development of an assay to assess PKC binding to several PH domains (Btk, dynamin, IR S l, Sos and Vav). GST-PH domain fusions were immobilised to glutathione sepharose beads and the matrix incubated with recombinant bacterial, affinity purified a, pi, pll, y

and C isoforms of PKC (kindly provided by Dr P. Parker; ICRF). After washing to remove non-specific interactions the protein complexes were resolved by SDS-PAGE, transferred to nitro-cellulose membrane and probed with the respective PKC isoform specific antibody. The binding of PKC isoforms to any of the test panel of PH domains was not detected. Moreover no PKC interaction could be seen with the PH domain of Btk (data not shown). The absence of a positive control to evaluate the PKC interactions resulted in the abandonment of the assay.

5.2.1.3 Py-subunits of heterotrimeric G-proteins

An insight into the potential function of PH domains came from the demonstration that the predicted PH domains of pARK and RasGRF extended by 30 amino-acid residues beyond the C-terminal domain boundary, could interact with Gpy subunits (Koch et al., 1993; Touhara et al., 1994). This led to a collaboration with Dr Gierschik (University of

Ulm, Germany) who used GST-fusion PH domains from pARK, Btk, dynamin, IRSl and Sos to competitively inhibit the in vitro Gpy mediated activation of recombinant PLC p2. In summary the competition assay was done in a total volume of 70 pi containing 0.3-1 pM isoprenylated Gpivi subunits, GST-PH domain fusion protein (0.3-1 pM), 28 pM

3[H]PtdIns(4,5)P2 (5 Ci/mol), 280 pM PtdEth, 50 mM Tris/HCL, pH 7.4, 2 mM EGTA, 80 mM KCl, 10 mM LiCl, 10 mM 2,3-bisphosphoglycerate, 1.2 mM sodium desoxycholate, and CaCl] to obtain 1 pM free Ca^+. PLC p2 was incubated with this

reaction mixture for 60 minutes at 30°C. Reactions were terminated by adding 350 pL CHCI3/CH3OH/HCl containing 5 mM EGTA (500:500:3). PLC p2 activity was measured

as radioactivity released into the upper aqueous phase (as quantitated by liquid- scindllation counting).

As well as using canonical PH domains in this assay, the analysis was also expanded to study the effect of C-terminally extended PH domains. pARK and Sos PH domains were originally expressed C-terminally extended. Dynamin and IRSl PH domains were C- terminally extended by 40 and 42 amino acids, respectively.

Affinity purification of IRSl and dynamin extended PH domains resulted in a homogenous and stable preparation of fusion proteins. Circular dichroism (CD) structural analyses of the thrombin cleaved dynamin extended PH domain showed a profile similar to that of the unextended domain. However there was an increase in contribution of random coil to the structure. CD spectra of the IRSl PH domains (canonical and extended) showed relatively little if any random coil. Comparison between the two 1RS 1 PH domains revealed only a slight increase in the character of antiparrallel p-sheet. These results are summarised in Figure 5.2. It should be emphasised that the scope of CD analysis in determining whether a protein is folded is limited, although it can accurately be used to ascertain if a small protein is predominantly folded.

Dr Gierschik's results showed that dynamin and C-terminally extended IRSl and pARK PH domains (all utilised as GST fusions in the assay) competitively inhibited the Gpy mediated stimulation of PLC p2 in a 1:1 stoichiometry. Inhibition of Gpy activation of PLC p2 was not observed with either GST-IRSl and -Btk PH domains or C-terminally extended Sos and dynamin GST fusion PH domains.

66— 55— 45— 36— 29— M 1 2 3 4

B

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Figure 5.2 CD analysis o f C-term inally extended dynam in and IR S l PH domains

A. Affinity purified C-term inally extended dynam in and IR S l PH domains.

Regions extending 38 and 42 residues, respectively from the C-terminiis of dynamin CFhr 511 -Met 670) and IRSl (Met 1-Gly 169) PH domains were expressed and affinity purified as GST- fusion proteins. For the purposes of CD structural analysis the GST tag was removed by thrombin digestion and the PH domains purified to homogeneity by FPLC. Lane I, GST-dynamin PH domain; lane 2, C-terminally extended GST-dynamin PH domain; lane 3, GST-lRSl PH domain; lane 4, GST-lRSl C-terminally extended PH domain. Lane M, molecular weight markers, kDa.

B. CD spectra from the analysis o f C -term inally extended dynam in and IR S l PH dom ains.

1. Typical CD spectra for various secondary structures, a-helix 1, antiparallel fLsheet 2, p tum 3, and random coil 4. The polypeptide backbone of L-amino acid residues is chiral and

gives an intense CD signal in the far-UV region (180-260 nm) that is dependent on the conformation of the backbone. In particular, a-helices, (3-sheets, and reverse turns each give characteristically different spectra in this wavelength range. The appropiate average content of such structures in a protein can be estimated from its CD spectrum, by comparison to a database of spectra of proteins of know n structure.

2. CD spectral comparison of the PH domains of dynamin (red) and C-terminally extended dynamin (blue).

3. CD spectral comparison of the PH domains 1RS 1 (ki^^ ), C-terminally extended 1RS 1 (black) and dynamin (red).