Dimeric PKD regulates membrane fission to form transport carriers at the TGN

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 01/2008; 179(6):1123-31. DOI: 10.1083/jcb.200703166
Source: PubMed


Protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) through interaction with diacylglycerol (DAG) and is required for the biogenesis of TGN to cell surface transport carriers. We now provide definitive evidence that PKD has a function in membrane fission. PKD depletion by siRNA inhibits trafficking from the TGN, whereas expression of a constitutively active PKD converts TGN into small vesicles. These findings demonstrate that PKD regulates membrane fission and this activity is used to control the size of transport carriers, and to prevent uncontrolled vesiculation of TGN during protein transport.

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    • "The cell-associated Tfn fluorescence was determined on a FACSVerse Flow Cytometer as EGFR-GFP and the data are shown as the relative values of MFI versus the initial value of each sample. HRP secretion assay ---HRP secretion assay was carried out as described previously (Bossard et al., 2007) with some modifications. Briefly, HepG2 cells were transfected with siRNA for -SNAP or -SNAP as described above. "
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    ABSTRACT: Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide sensitive factor (NSF) and its attachment protein, α-SNAP, catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of the fusion event. γ-SNAP is an isoform of SNAP, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates endosomal trafficking of epidermal growth factor receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry revealed that γ-SNAP interacts with limited SNAREs including endosomal ones. γ-SNAP, as well as α-SNAP, mediated disassembly of endosomal syntaxin 7-containing SNARE complexes. Overexpression and small interfering RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in limited organelles including endosomes and their trafficking pathways. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 06/2015; 128(15). DOI:10.1242/jcs.158634 · 5.43 Impact Factor
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    • "Another level of complexity in regulation of cofilin activity is achieved because PKD enzymes can form protein complexes (Fig. 1 and [26]). To simplify our studies we used two cell lines as a model system that only express PKD2 and PKD3. "
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    ABSTRACT: Background Protein kinase D (PKD) enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4) and the phosphatase slingshot 1L (SSH1L). The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently. Methodology/Principal Findings We here analyzed two cell lines (HeLa and MDA-MB-468) that express the subtypes protein kinase D2 (PKD2) and protein kinase D3 (PKD3). We show that under normal growth conditions both isoforms can form a complex, in which PKD3 is basally-active and PKD2 is inactive. Basal activity of PKD3 mediates PAK4 activity and downstream signaling, but does not significantly inhibit SSH1L. This signaling constellation was required for facilitating directed cell migration. Activation of PKD2 and further increase of PKD3 activity leads to additional phosphorylation and inhibition of endogenous SSH1L. Net effect is a dramatic increase in phospho-cofilin and a decrease in cell migration, since now both PAK4 and SSH1L are regulated by the active PKD2/PKD3 complex. Conclusions/Significance Our data suggest that PKD complexes provide an interface for both cofilin regulatory pathways. Dependent on the activity of involved PKD enzymes signaling can be balanced to guarantee a functional cofilin activity cycle and increase cell migration, or imbalanced to decrease cell migration. Our data also provide an explanation of how PKD isoforms mediate different effects on directed cell migration.
    PLoS ONE 05/2014; 9(5):e98090. DOI:10.1371/journal.pone.0098090 · 3.23 Impact Factor
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    • "PKC in concert with ARF regulates rat brain PLD (Kanaho et al. 1996), and the addition of GTPcS potentiates PKC-mediated PLD activation (Ohguchi et al. 1995). PKD binds to the TGN and participates in the formation of post-Golgi transport carriers (Liljedahl et al. 2001; Bossard et al. 2007). The first of the PKD cysteine-rich domains (C1a), but not the second (C1b), is sufficient for the binding of PKD to the TGN (Maeda et al. 2001), which occurs in a DAG-dependent manner (Baron and Malhotra 2002). "
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    ABSTRACT: Intracellular membrane transport involves the well-coordinated engagement of a series of organelles and molecular machineries that ensure that proteins are delivered to their correct cellular locations according to their function. To maintain the homeostasis of the secretory system, the fluxes of membranes and protein across the transport compartments must be precisely balanced. This control should rely on a mechanism that senses the movement of the traffic and generates the required homeostatic response. Due to its central position in the secretory pathway and to the large amounts of signaling molecules associated with it, the Golgi complex represents the ideal candidate for this regulation. The generation of autonomous signaling by the Golgi complex in response to the arrival of cargo from the endoplasmic reticulum (ER) has been experimentally addressed only in recent years. These studies have revealed that cargo moving from the ER to the Golgi activates a series of signaling pathways, the functional significance of which appears to be to maintain the homeostasis of the Golgi complex and to activate Golgi trafficking according to internal demand. We have termed this regulatory mechanism the Golgi control system. A key player in this Golgi control system is the KDEL receptor, which has previously been shown to retrieve chaperones back to the endoplasmic reticulum and more recently to behave as a signaling receptor. Here, we discuss the particular role of KDEL receptor signaling in the regulation of important pathways involved in the maintenance of the homeostasis of the transport apparatus, and in particular, of the Golgi complex.
    Histochemie 07/2013; 140(4). DOI:10.1007/s00418-013-1130-9 · 3.05 Impact Factor
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