Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin

Abstract

We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.The mechanistic elucidation of signaling pathways regulating dynamic actin remodeling processes in migrating cells is pivotal to a comprehensive understanding of cancer cell metastasis. Protein kinase D (PKD) 2 has recently been identified as a vital upstream regulator of polarized cell motility and F-actin organization (1-4). PKD localizes to sites of dynamic actin remodeling (1). The kinase activity is essential for the control of directed cell motility (1, 2), whereby active PKD1 inhibited, whereas kinase-inactive PKD1KD strongly enhanced motility and invasiveness (1-4). Mechanistically, a key role for PKD1 in controlling the activity of the ubiquitous F-actin depolymerizing-and severing factor cofilin via slingshot1L (SSH1L) cofilin phosphatase has been demonstrated. The activity of SSH1L is mainly regulated by its binding to filamentous actin (F-actin), which has been shown to strongly enhance its activity (5, 6). Phosphorylation of SSH1L at Ser 978 by active PKD1, e.g. downstream of RhoA or oxidative stress, generates a 14-3-3 binding motif within an important F-actin binding region, thus resulting in the sequestration of SSH1L away from dynamic actin structures, reducing SSH1 activity and active non-S3-phosphorylated cofilin levels (2). By severing actin filaments, cofilin increases both the availability of G-actin monomers as well as the number of "barbed ends" for polymerization (7). Furthermore, severed filaments are the preferred substrate for dendritic nucleation by the Arp complex (8, 9). Localized induction of actin polymerization and the formation of branched actin networks constitute the basis for membrane protrusion and cell motility (2, 10, 11). In line with an upstream regulatory role in the control of these processes, PKD1 and -2 are also capable of binding to F-actin in vitro (1). In the case of PKD1, in vivo F-actin binding has been demonstrated as well, which most likely facilitates an interaction with actin regulatory proteins such as SSH1L (2). We now have identified a second key regulatory signaling pathway ...

GIT1 Phosphorylation on Serine 46 by PKD3 Regulates Paxillin Trafficking and Cellular Protrusive Activity

“…At the Golgi complex, PKD regulates vesicular traffic to the plasma membrane, whereas at the plasma membrane PKD is involved in the regulation of cell shape, movement, and invasion. In line with such functions, substrates associated with actin cytoskeletal remodeling including cortactin, DLC1, RIN1, SSH1L, and Pak4 were recently identified to be phosphorylated by PKD (21)(22)(23)(24)(25)(26). Most of these studies focused on PKD1 and/or PKD2 isoforms, whereas relatively little is known about PKD3.…”

Protein kinase D in vascular biology and angiogenesis

“…This may be an important goal for future therapeutics as there is emerging evidence that PKD isoforms play different roles in tumors of different origin. For example, a series of studies by Eiseler and coworkers found that PKD1 negatively regulates migration of Panc89 cells through several related mechanisms including association of PKD with F-actin and phosphorylation of cortactin and slingshot 1 (SSH1) (42)(43)(44). In addition, PKD1 expression is decreased in invasive breast cancers and expression of a constitutively active PKD1 mutant decreased the invasiveness of MDA-MB-231 breast carcinoma cells by causing a subsequent decrease in matrix metalloproteinase expression (45).…”

Control of MT1-MMP transport by atypical PKC during breast-cancer progression

“…We tested whether cortactin was a direct substrate for aPKCζ/ι by incubating purified GST-tagged human cortactin in the presence of recombinant human aPKCι. Phosphorylation was detected with antibodies raised against cortactin phosphoSer298, which was recently identified as a phosphorylation site for aPKCζ/ι-related PKD (35,36). In higher metazoans, mRNA splicing generates cortactin variants with four, five, or six conserved tandem actin-binding repeats in which repeats 5 and 6 are the most conserved (37).…”