PKD regulates membrane fission to generate TGN to cell surface transport carriers

Abstract

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) by binding diacylglycerol (DAG) and the ARF1 GTPase. PKD, at the TGN, promotes the production of phosphatidylinositol-4 phosphate (PI4P) by activating the lipid kinase phophatidylinositol 4-kinase IIIß (PI4KIIIß). PI4P recruits proteins such as oxysterol-binding protein 1 (OSBP) and ceramide transport protein (CERT) that control sphingolipid and sterol levels at the TGN. CERT mediated transport of ceramide to the TGN, we suggest, is used for increasing the local production and concentration of DAG. Once the crucial concentration of DAG is achieved, OSBP and CERT dissociate from the TGN on phosphorylation by PKD and DAG is sequentially converted into phosphatidic acid (PA) and lyso-PA (LPA). Therefore, the net effect of the activated PKD at the TGN is the sequential production of the modified lipids DAG, PA, and LPA that are necessary for membrane fission to generate cell surface specific transport carriers.

Figure 1.

Exit routes for cargo at the Golgi. Although all proteins that exit the ER are transported by COPII vesicles to the ERGIC, there are numerous exit routes for cargo at the TGN. Traffic to the endosomes is mediated by clathrin coated vesicles; however, the molecular composition of transport carriers departing from the TGN to other cellular destinations is still unclear. Not all cells have the same exit routes and there are likely to be additional cell type dependent routes for cargo export from the Golgi. Do all transport vesicles that form at the TGN use the same fission components?

Figure 2.

Ilimaquinone (IQ) cuts the entire Golgi apparatus to produce uniform size vesicles. The sponge natural metabolite Ilimaquinone vesiculates the Golgi apparatus by overactivating the membrane fission reaction. IQ-mediated Golgi vesiculation requires trimeric G protein and PKD. Surprisingly, however, PKD is required only for the fission of transport carriers containing the basolateral surface specific cargo at the TGN.

Figure 3.

Recruitment of PKD to the TGN. (A) The known domains of PKD. The PKD family in mammals comprises three members: PKD1, PKD2, and PKD3. All known members share a highly conserved amino-terminal regulatory domain, which is composed of two cysteine-rich domains (C1a and C1b) and an auto-inhibitory PH domain in addition to the catalytic kinase domain. PKD1 and PKD2 contain an amino-terminal hydrophobic stretch of amino acids (P), which we suggest penetrates the outer leaflet of the TGN. (B) The C1a domain of PKD binds DAG via proline 155. Proline 275 in the C1b domain of PKD is required for binding ARF1 at the TGN. These two domains in addition to the amino-terminal hydrophobic patch anchor PKD to the TGN. DAG bound to the C1a domain cannot flip across the bilayer and this is important, we suggest, to concentrate DAG in the outer leafter for events leading to membrane fission. ARF1 bound to the C1b domain of PKD cannot recruit COPI coats and we propose is required for the activation of PLD.

Figure 4.

Activation of PKD. The PH domain of PKD is known to be a negative regulator of its kinase activity. Binding of Gßγ would alleviate the negative effect of the PH on the kinase activity of PKD. The MAPK p38δ phosphorylates in the PH domain to inactivate PKD. PKCη is an activator of PKD. In addition, Ca²⁺ and DAG also control the activity and the duration of the kinase activity of PKD.

Figure 5.

The effectors of PKD at the TGN. (A) PKD is recruited to the TGN by DAG and ARF1. Activated PKD phosphorylates to activate PI4KIIIß thus promoting the production of PI4P in the outer leaflet of the TGN. (B) PI4P recruits a number of proteins to the TGN membrane, including OSBP and CERT. (C) OSBP and CERT regulate the sphingomyelin and sterol levels in the TGN and this we suggest is required for separating the PI4P containing domain from the SM and sterol rich domain. CERT dependent ceramide transport to the TGN, we suggest is required to generate and concentrate DAG at the TGN. (D) This DAG in turn recruits more PKD to generate more DAG. PKD then phosphorylates OSBP and CERT to release them from the TGN. (E) ARF1 mediated activation of PLD1 generates PA from the PC pool, which eventually is converted to LPA by the action of PLA2. The accumulation of these different modified lipids leads to fission of TGN to cell surface transport carriers. (F) DAG is consumed thus dissociating PKD from the membrane thus resetting the TGN to generate another round of transport carriers in a cargo-dependent manner.