PKDs play a major role in regulating protein transport from the TGN to the plasma membrane. There has been a substantial amount of work elucidating the mechanisms by which PKDs regulate vesicle shedding. A local pool of DAG plays a major role in the recruitment of PKD to the TGN (Bard and Malhotra, 2006
; Bossard et al., 2007
). Active PKD phosphorylates PI4KIIIβ and CERT, the two important PKD substrates identified so far at the TGN. The PKD-mediated phosphorylation of PI4KIIIβ and CERT is critical in regulating the cross-talk between the membrane lipid biogenesis and protein secretion. This in turn regulates the maintenance of local DAG and PKD tethering to the TGN, which ultimately ensures a controlled vesicular transport process from the TGN (Hausser et al., 2005
; Bard and Malhotra, 2006
; Fugmann et al., 2007
). Previous work demonstrated that the C1a domain of PKDs is crucial for the localization of PKDs at the TGN via binding of DAG (Maeda et al., 2001
). However, the precise mechanisms how PKDs are recruited to the TGN are as yet less clear.
The small GTPases of the ADP-ribosylation factor family are also master regulators of the structure and function of the Golgi complex. Among the three classes of the ARF family, class I and II were reported to exert their function at the Golgi compartment. Active ARF1 recruits COPI coats which interact with the bona fide cargo proteins and generate functional vesicles that operate in the intra-Golgi and Golgi-endoplasmic reticulum retrograde trafficking zones of the membrane trafficking process (Orci et al., 1993
). In addition, ARF1 was also shown to be one of the major components of the sorting machinery and is involved in controlling multiple TGN exit pathways (De Matteis and Luini, 2008
). Many of the effectors and regulators of ARF1 play an important role in the formation and scission of vesicles destined for distinct compartments of the cell.
Here, we demonstrate that PKD2 specifically and directly interacts with ARF1. Binding of PKD2 to ARF1 is affected by the nature of the nucleotide bound to the GTPase and the association of PKD2 with ARF1 is enhanced when the GTPase exists in active confirmation. However, there is also an interaction between PKD2 and inactive ARF1. This is in line with previous reports that described the association of ARF1 with effector proteins such as the HIV Nef protein and the μ subunit of the adaptor protein complex AP-4 that interact with ARF1 independently of the nucleotide status of the GTPase (Boehm et al., 2001
; Faure et al., 2004
Expression of dominant-negative mutants of ARFs that are locked in the GDP conformation serve as an important tool in studying the effect of different ARF isoforms on the subcellular localization of various effector proteins. We found that the localization of PKD2 at the TGN was regulated by class I and II ARFs, which are known to play indistinguishable roles at the Golgi complex.
ARF1 and PKD2 not only interact in vitro and in vivo but also colocalize at the TGN as demonstrated by immunocytochemistry. In addition, the interaction of PKD2 with ARF1 is specifically mediated by its C1b domain and Pro275 within this domain is the central amino acid required for the ARF1-PKD2 interaction. A PKD2 mutant lacking the C1b domain or exhibiting a P275G exchange not only fails to interact with ARF1 but also does not localize to the Golgi and is largely localized in the cytoplasm. This points to a crucial role for the interaction of PKD2 with ARF1 to target PKD2 to the Golgi compartment. Our study shows that ARF1 functions as an important receptor for PKD in addition to local pool of DAG at the TGN. This also explains the requirement of an additional mechanism to target PKD to the TGN, despite the fact that DAG is present at various cellular locations. In line with this conclusion, our data further demonstrate that the loss of the C1b domain or the P275G exchange abolish the functional activity of PKD2 at the Golgi compartment. Both mutants cannot rescue the block of protein transport from the TGN to the plasma membrane induced by siRNA-mediated knockdown of endogenous PKD2 and -3 in HeLa cells. Furthermore, a lack of the C1b domain or a P275G mutation also abolished the dominant-negative effect of kinase dead PKD2 on protein transport from the TGN.
In conclusion, these data suggest a novel model in which the localization of PKD2 to the TGN requires both, the C1a and C1b domain: ARF1 recruits PKD2 from the cytoplasm (a) to the Golgi apparatus via binding of Pro275 in the C1b domain of PKD2. The kinase is then anchored at the TGN by interacting with DAG in the membrane via its C1a domain. Both processes are required to accomplish vesicle shedding (b). When the C1b domain is deleted or the critical Pro275 in PKD2 is mutated, ARF1 binding is impaired resulting in cytoplasmic localization of PKD2 and loss of function at the Golgi (c). These data provide the first link between the “classical” machinery regulating protein transport at the Golgi compartment, namely, ARF proteins, and PKDs and demonstrate that the direct interaction of both is crucial for efficient protein transport from the TGN to the plasma membrane.
Figure 7. Model depicting PKD2 recruitment and function at the TGN. PKD2 localized in the cytoplasm (a) is recruited to the TGN by binding to ARF1 via Pro275 within the C1b domain. This results in further positioning by interaction with DAG via C1a domain and thereby (more ...)