Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA

SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2 purinoceptor through which UTP, ATP, and ATPγS stimulate secretion with EC50 values of ∼3 μM. PMA elicits mucin secretion with high EC50 (75 nM) and saturation (300 nM) values. For the first time in airway mucin-secreting cells, the PKC isoforms expressed and activated by a secretagogue were determined using RT-PCR/restriction-enzyme mapping and Western blotting. Five isoforms were expressed: cPKCα, nPKCδ and -η, and aPKCζ and -ι/λ. PMA caused cPKCα and nPKCδ to translocate to the membrane fraction of SPOC1 cells; only nPKCδ so responded to ATPγS. Membrane-associated nPKCδ and mucin secretion increased in parallel with ATPγS concentration and yielded EC50 values of 2–3 μM and maximum values of 100 μM. Effects of PMA to increase membrane-associated cPKCα and nPKCδ saturated at 30 nM, whereas mucin secretion saturated at 300 nM, which suggests a significant PKC-independent effect of PMA on mucin secretion. A prime alternate phorbol ester-receptor candidate is the C1-domain protein MUNC13. RT-PCR revealed the expression of ubiquitous (ub)MUNC13-2 and its binding partner, DOC2-γ. Hence, P2Y2 agonists activate nPKCδ in SPOC1 cells. PMA activates cPKCα and nPKCδ at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.