Marco conceptual

Initial experiments to determine if phorbol esters could synergize with PS and PA to modulate «-chimaerin were performed with saturating concentrations i.e. 1 0 0 pg/ml

o f the two phospholipids. This entailed pre-incubation of n-chimaerin with either phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate in the presence o f PS and PA (100 pg/ml) prior to the determination of the t^Q% for R acl.G TP. No additional stimulation by phorbol esters apart from that attributed to the phospholipids was observed. To optimize the condition for synergism, limiting PA and PS concentrations (5 pg/ml) were next employed. Table 4 shows that under these conditions, synergism between phospholipids and phorbol esters was observed. The largest decrease in was seen with the PA/PMA combination. Stimulation by PMA was dependent on PS or PA. aPM A, an inactive stereoisomer of PMA that does not bind or regulate PKC, was ineffective. Addition of the solvent previously used to solubilize phorbol esters (i.e. acetonitrile/water, 2:3, v/v) to either PS or PA did not affect R acl GAP activity of /i-chimaerin. Furthermore, in the absence of PS and PA, 100 nM of both PMA and PDBu did not affect the intrinsic rate of GTP hydrolysis by R acl. Unlike phorbol esters, diacylglycerol (DAG) did not synergize with PS or PA at either low or high concentration of the phospholipid. This inability o f DAG to synergize with PS or PA might be explained by the fact that suboptimal conditions were used and does not rule out a potential effect o f DAG on n-chimaerin. Further work, including the use of eukaryotically expressed or native R acl and n-chimaerin proteins, will be necessary to determine this possibility. It was also observed that LPA at 258 pg/ml reversed the stimulation seen with either PA (100 pg/ml) or PS (5 pg/ml)/PMA (100 nM) (Table 5).

3.5 /i-chimaerin physically interacts with phospholipids

To examine whether physical interaction with phospholipids was sufficient for modulation of the R acl GAP activity of n-chimaerin, a sedimentation assay was employed. Liposomes of different phospholipid composition (PC, PE, PS, PA or LPA) were intially incubated for 1 h at room temperature with GST fusion proteins containing the full-length and carboxyl-terminal region of n-chimaerin. This was

Table 4 Synergism between phospholipids and phorbol esters in activating n-

chimaeiin

The R acl GAP activity o f «-chimaerin (125 ng) was measured in the presence o f 5 pg/ml phosphatidylserine (PS) or phosphatidic acid (PA) together with 100 nM phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate (PMA/PDBu) following a 30 min pre-incubation with the stipulated combination o f lipids. Addition o f solvent used to dissolve phorbol esters (acetonitrile/water, 2:3, v/v) to either PS or PA did not affect R acl GAP activity o f «-chimaerin. Data below are averages ± S.D. from three separate experiments.

Addition ^50% (lïiin) None 11.9 ± 0 . 1 «-chimaerin 1 0 . 0 ± 0 . 0 «-chimaerin/PA 10.3 ± 0.4 «-chimaerin/P A/PDB u 8.7 ± 0.1 «-chimaerin/PS 9.9 ± 0 . 1 n -chimaerin/PS/PDB u 8 . 8 ± 0 . 6 «-chimaerin/PS/PM A 8 . 6 ± 0 . 2

Table 5 Stimulatoiy effects of /i-chimaerin Racl GAP activity by phospholipids and phorbol ester are inhibited by lysophosphatidic acid

The R acl GAP activity of n-chimaerin was measured in the presence of: (a) 100 pg/ml lysophosphatidic acid (LPA), 5 pg/ml phosphatidylserine (PS) and 100 nM phorbol 12- myristate 13-acetate (PMA); (b) 100 pg/ml phosphatidic acid (PA) and either 100 pg/ml or 200 pg/ml lysophosphatidic acid. 250 ng and 125 ng n-chimaerin protein were used in (a) and (b) respectively. In both experiments, incubation of the R acl GAP assay mixture was for 10 min at 15°C before membrane filtration. R acl GAP activity is presented as the % GTP remaining bound to R acl under various conditions stipulated below. Data below are averages ± S.D. from a duplicate experiment. In the absence of n-chimaerin, additions of the solvent used to dissolve PMA (acetonitrile/water, 2:3, v/v) as well as any of the above lipids did not affect the intrinsic GTPase activity of R acl.

(a)

Addition % GTP remaining after 10 min

None 73.5 ± 0.6 «-chimaerin 43.7 ± 1.2 w-chimaerin/LPA 59.6 ± 0.4 «-chimaerin/PS/PM A 34.5 ± 0.8 n -chimaerin/LP A/PS/PM A 53.3 ± 1.4 (b)

Addition % GTP remaining after 10 min

None 71.5 ± 1.4

«-chimaerin 58.2 ± 1.1

«-chimaerin/PA 27.5 ± 0.5

«-chimaerin/100 |ig/ml LPA 66.3 ± 0.5

«-chimaerin/PA/200 pg/ml LPA 67.7 ± 0.2

Fig. 23 A^-chimaerin physically interacts with phospholipids via its PKC-like cysteine-rich domain

The partitioning of full-length n-chimaerin into the supernatant (=) and liposomes (■) were examined using liposome co-sedimentation technique (see section 6.3 of the M aterials and Methods chapter). This was performed either (1) in the absence or presence o f the following liposomes: (2) lysophosphatidic acid, (3) phosphatidic acid, (4) phosphatidylserine, (5) phosphatidylcholine and (6) phosphatidylethanolamine. No

carboxyl-terminal n-chimaerin protein co-sedimented with the liposome and hence did not physically interact with the liposomes from any phospholipids tested. Similar results were obtained in two separate experiments.

% protein

a\ o\

followed by centrifugation allowing separation into two phases. Full-length n- chimaerin fusion protein sedimented with liposomes formed from all the phospholipids tested except PE (Fig. 23). Neither GST nor the GST/carboxyl-terminal region protein sedimented with liposomes of any composition. It was noted that although PC physically interacted, it did not affect n-chimaerin activity (Fig. 18). Thus, physical interaction with phospholipids is not sufficient for modulation o f the R acl GAP activity o f n-chimaerin and it is likely that more specific interactions, as determined by the chemical structure of the phospholipid, are required.

3.6 Sphingosine inhibits the Racl GAP activity of both full-length and caiboxyl-