The PID Regulatory Complex

Several PID regulatory proteins have been identified in a yeast two-hybrid screen as interactors of PID. Two of them, PINOID Binding Protein 1 (PBP1)

and TOUCH3 (TCH3), are calmodulins that show calcium-dependent binding to PID and respectively enhance and downregulate PID activity in vitro (Fig. 2). Neither PBP1 nor TCH3 seem to be phosphorylation targets of PID, and assays in which 35S::PID seedlings were treated with calcium transporter and calmodulin inhibitors showed enhanced root meristem collapse (Benjamins et al. 2003). In addition, we have obtained further data that support a role for PBP1 and TCH3 as regulators of the activity and subcellular localization of the PID kinase in vivo (H. Robert et al., unpublished data).

A third interactor and regulator of PID is the PINOID Binding Protein 2 (PBP2). PBP2 contains two protein-protein interaction domains, the BTB/POZ- (Bric-a-brac, Tramtrack and Broad Complex/Pox virus and Zinc finger) and the TAZ (Transcriptional Adaptor putative Zinc Finger) domain. PBP2 has previously been identified as a calmodulin binding transcriptional regulator AtBT1 (Du and Poovaiah 2004). Our observations suggest that PBP2 acts as an inhibitor of PID, since it represses PID auto- and trans-phosphorylation activity in vitro (Benjamins 2004; Zago 2006). Moreover, the fact that the GFP-PBP2 fusion protein shows a cytoskeleton-like localization in onion cells (Benjamins 2004), suggests that PBP2 provides a possible link between the established roles of PID and the cytoskeleton in regulating PAT. Although the subcellular localization of PBP2-GFP in Arabidopsis protoplasts and plants showed nuclear and general cytosolic rather than cytoskeleton localization, a yeast two-hybrid screen identified two closely related microtubule motors (kinesins) as interacting partners of PBP2, which were named PBP2 Binding Kinesin 1 and 2 (PBK1 and 2). Preliminary data from both in vitro and in vivo experiments corroborate the involvement of the PBKs in the PID signaling pathway. On the basis of these observations, we speculate that the PBKs, together with PBP2, are involved in repression, and possibly determine the subcellular localization of PID activity (Zago 2006).

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