Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Abstract

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation

“…β-Catenin-GST and vinculin-His 6 were expressed in BL21-bacteria and purified as described previously (Eiseler et al, 2016). For pulldown experiments, 20 µl of purified protein on beads was incubated for 4 h with 2 mg of total cell lysate from HEK293T cells expressing the indicated binding partners.…”

“…For pulldown experiments, 20 µl of purified protein on beads was incubated for 4 h with 2 mg of total cell lysate from HEK293T cells expressing the indicated binding partners. Pulldown assays were performed as described previously (Eiseler et al, 2016). Empty beads and GST-conjugated protein beads were used as negative controls.…”

“…This method allows the determination of protein-protein interactions indicated by an apparent molecular proximity below 10 nm (Eiseler et al, 2012(Eiseler et al, , 2016Gu et al, 2004;Kunkel and Newton, 2009;Oser et al, 2010;Valente et al, 2012;Wille et al, 2014;Wouters et al, 1998;Zeug et al, 2012). The FRET acceptor in a region of interest (ROI) was bleached by an intensive laser line and increase in donor fluorescence, indicating FRET, was measured.…”

Multiple activities of Arl1 GTPase in the trans-Golgi network

“…Genetic alterations of Arl1 as well as its effectors were used to characterize developmental and physiological phenotypes (summarized in Fig. 1) (Chang et al, 2015;Cruz-Garcia et al, 2013;Eiseler et al, 2016;Gehart et al, 2012;Hesse et al, 2013;Hsu et al, 2016;Labbaoui et al, 2017;Lieu et al, 2008;Liu et al, 2006;Lock et al, 2005;Marešová and Sychrová, 2010;Marešová et al, 2012;Murray and Stow, 2014;Price et al, 2005;Torres et al, 2014;Yang and Rosenwald, 2016). In mammalian cells, these functions affect a wide range of fundamental cellular processes, including cell polarity (Lock et al, 2005), innate immunity (Bremond et al, 2009;Lieu et al, 2008;Murray and Stow, 2014;Stanley et al, 2012), lipid droplet and chylomicron formation (Hesse et al, 2013;Jaschke et al, 2012), as well as the secretion of insulin (Gehart et al, 2012), chromogranin A (CruzGarcia et al, 2013) and matrix metalloproteinases (MMPs) (Eiseler et al, 2016).…”

Role of Signal-Regulated Changes in Microtubule Stability Dynamics through <i>α</i>-Tubulin Phosphorylation on Ser/Tyr in Modulation of Salivary Gland Matrix Metalloproteinase-9 (MMP-9) Secretion in Response to <i>Porphyromonas gingivalis</i> and Ghrelin

“…While GDP-bound inactive Arfs are cytosolic, the stimulus activated GTP-bound class I Arfs (Arf1, Arf2, and Arf3) rapidly translocate to the Golgi membrane compartments and assume the principal role in the recruitment of various cytosolic coat and cargo adaptor proteins, exchange factors, and lipid modifying enzymes that are essential for regulation of ER-to-Golgi traffic [11] [12]. Of particular significance to the MMP-9-containing secretory cargo processing is the role of Arf1 in the recruitment from the cytoplasm to the TGN of PKD2 [14] [15]. The PKD family of serine/threonine kinases (PKD1, PKD2, and PKD3), plays a crucial role in the regulation of Golgi structure and function by phosphorylation of the TGN-localized substrates required for subsequent shedding of cargo-containing vesicles [10].…”