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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.

SUBMITTER: Eiseler T

PROVIDER: S-EPMC2881792 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin.

Eiseler Tim T Hausser Angelika A De Kimpe Line L Van Lint Johan J Pfizenmaier Klaus K

The Journal of biological chemistry 20100402 24

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, cortact ...[more]

PMID: 20363754

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Project description:Malregulation of the actin cytoskeleton enhances tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin-related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of cortactin T24 by CK2α impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. IMPLICATIONS: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.

Project description:The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

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Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin.

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Protein kinase D controls actin polymerization and cell motility through phosphorylation of cortactin.

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