AtPIP2;1 facilitated ion transport is influenced by activity of endogenous oocyte kinases
Stimulation or inhibition of the endogenous kinase activity of X. laevis oocytes expressing wild-type (WT) AtPIP2;1 was manipulated by treatment with cyclic nucleotide monophostphates (cNMPs) or a kinase inhibitor (H7). These treatments resulted in changes in the magnitude of AtPIP2;1-associated ionic conductance when compared to untreated AtPIP2;1 or either treated or untreated water injected control oocytes (Figure 1). Treatments with cAMP and cGMP can stimulate oocyte endogenous kinase activity, and this can alter protein phosphorylation (Glass and Krebs, 1980; Kuwahara et al., 1995). Different kinases respond to cAMP verses cGMP signalling, and the kinases responding to these different signals have different target sites (Conti et al., 2012). Endogenous oocyte kinases have been previously shown to alter plant aquaporin phosphorylation state and influence their water channel activity, and this was demonstrated by applying treatments with kinase stimulators and inhibitors (Maurel et al., 1995; Van Wilder et al., 2008). Treatment of AtPIP2;1 expressing oocytes with cAMP or cGMP increased and decreased AtPIP2;1-associated ionic conductance, respectively (Figure 1). Treatment of AtPIP2;1 expressing oocytes with the kinase inhibitor H7 decreased AtPIP2;1-associated ionic conductance (Figure 1b). H7 treatment similarly reduced aquaporin-associated ionic conductance for HsAQP1 expressing oocytes by inhibiting the influence of cAMP on endogenous kinases (Yool et al., 1996). The different responses to the different cNMP treatments indicate that multiple phosphorylation sites, such as candidate sites in the CTD may be involved in regulating AtPIP2;1-facilitated ionic conductance and that transport function can be altered by the activity of endogenous oocyte kinases. However, a direct effect of cNMPs on AtPIP2;1 may also be possible; HsAQP1 ion channel function is activated by direct cGMP binding to its loop D in a phosphorylation-dependent manner (Anthony et al., 2000; Campbell et al., 2012) although an equivalent site is not present in AtPIP2;1.