Discusión sobre el cálculo de la tensión de borde

The data presented so far in this chapter demonstrates that PKD transiently localises to the plasma membrane of B cells and mast cells in response to antigen receptor ligation before subsequently returning to the cytosol, within 10 minutes of the initiation of antigen receptor signalling. One question that was important to address was whether dissociation of PKD from the plasma membrane was accompanied by a reduction in its kinase activity. The data presented in Chapter 5 indicated that substantial PKD activity following antigen receptor engagement in both B cells and in mast cells could be detected

at times when PKD dissociation from the plasma membrane was essentially complete (this chapter).

To investigate the long-term kinetics of PKD activation by antigen receptors, A20 B cells were stimulated with F(ab)’2 fragments of anti-mouse IgG, to stimulate the BCR, for a period of up to 2 hours. PKD activity was then measured at the indicated time points by in vitro kinase assays and also by western blot analysis of total cell lysates using the pS916 antibody (which selectively recognises active PKD). As demonstrated in Figure 6.13, BCR-induced activation of PKD in B cells was an extremely sustained response: maximal activity was observed within 1 minute of antigen receptor ligation and this was maintained at high levels throughout the 10 minute period that PKD was found associated with the plasma membrane in confocal microscopy experiments. Importantly, both PKD catalytic activity and S916 phosphorylation remained high for at least two hours post-receptor triggering, at times when PKD had fully dissociated from the plasma membrane (compare Fig. 6.7 and Fig. 6.13).

The confocal images presented in Figure 6.7 revealed that PKD does not appear to localise to specific intracellular structures following its dissociation from the plasma membrane of antigen receptor-stimulated cells. To explore the subcellular localisation o f active PKD further, cytosolic and total membrane fractions were prepared from BCR- stimulated A20 B cells. Western blot analysis of these fractions, using the pS916 antibody to identify activated PKD, revealed that active PKD could be found localised to the particulate (membrane) fraction of these cells within 2 minutes of BCR-stimulation but within ~15 minutes PKD activity within this compartment had diminished (Fig. 6.14a). In contrast, active PKD was detectable within the soluble (cytosolic) compartment of B cells up to 30 min after BCR engagement (Fig. 6.14a), indicating that active PKD does not strongly associate with intracellular membranes following its dissociation from the plasma membrane. Importantly, only PKD isolated from membrane fractions of PDBu-treated B cells showed immunoreactivity with the pS916 antiserum. Moreover, prolonged stimulation of PKD with phorbol esters (>1 h) resulted a sustained association of active PKD with cellular membranes (Fig. 6.14b). This was in agreement with the confocal data presented in this chapter demonstrating that phorbol esters induce the sustained plasma membrane localisation of GFP-PKD in intact cells. Accordingly, these confocal images and biochemical data indicate that there are two phases of PKD regulation following antigen receptor stimulation in B cells and in mast cells: an initial activation/recruitment to the plasma membrane followed by a sustained period of PKD activity within the cytosol.

n/s 2 15 30 60 120 min

IV K

a - p S 9 1 6

a -P K D

Fig. 6.13. Sustained PKD activity following BCR ligation - I.

A2Ü B lymphocytes were plated in complete medium at a density o f 1.5x10”^ cells ml in a 24 well plate and left to recover at 37°C for 1 h before stimulation. Subsequently the cells were left untreated (n s) or were stimulated with IÜ /./g ml F(ab)'2 fragments o f anti-mouse IgG for various times (2-120 min). The cells were lysed and PKD immunoprecipitated using the PA-1 antiserum. In vitro kinase assays (IV K ) were the performed using PKD autophosphorylation as the measure o f catalytic activity. Alternatively, proteins in the lysates were precipitated w ith cold acetone, separated by SDS-PAGE and subjected to western blot analysis using the pS9I6 antiserum and a pan C-terminal PKD antibody (sc-935). Results are representative o f 3 independent experiments.

Fab’2 2 5 15 30 PDBu 10 min F ab ’2 2 5 15 30 m in a-pS9l6

B)

Fig. 6.14. Sustained PKD a ctivity follow ing BCR ligation - II.

(A) A2Ü B lymphocytes were left untreated (-), treated w ith 50 ng ml PDBu for 10 min or were stimulated with 10 /fg ml F(ab)'2 fragments o f anti-mouse IgG for various times (2-30 min). Particulate (P; membrane) and soluble (S; cytosolic) fractions were prepared from the cells, as described in Chapter 2. Total proteins in the resulting fractions were precipitated w ith cold acetone, separated by SDS-PAGE and subjected to western blot analysis using the pSO 16 antiserum to detect active PKD (left panel) before reprobing w ith a pan erk 2 antibody (right panel). Results are representative of 2 independent e.xperiments.

(B) PKD activity is associated with cellular membranes in a sustained manner in phorbol ester- stimulated cells. COS-7 cells e.xpressing wild-type PKD were left unstimulated (0) or were treated with 50 ng ml PDBu for 15 or 60 min. Cells were fractionated as described in Chapter 2 and the fractions analysed by SDS-PAGE and western blotting with the pSO 16 antisernm to detect active PKD followed by a pan C-terminal PKD antibody (sc-935). Data are representative o f 3 independent e.\periments.

6.3. DISCUSSION.

The data presented in this chapter uses a combination of biochemical and confocal microscopy techniques to reveal spatial and temporal aspects of PKD regulation. There has been no previous analysis of the localisation of PKD during a physiological response in lymphocytes and in particular no analysis of the subcellular distribution of active PKD in vivo. The data presented in this chapter has explored PKD activity and subcellular localisation in response to both pharmacological (phorbol esters) and physiological (antigen receptors) stimuli.