Elevated protein kinase D3 (PKD3) expression supports proliferation of triple-negative breast cancer cells and contributes to mTORC1-S6K1 pathway activation.

Here, we show that the expression of the Golgi-localized serine-threonine kinase protein kinase D3 (PKD3) is elevated in triple-negative breast cancer (TNBC). Using an antibody array, we identified PKD3 to trigger the activation of S6 kinase 1 (S6K1), a main downstream target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Accordingly, PKD3 knockdown in TNBC cells led to reduced S6K1 phosphorylation, which was associated with impaired activation of mTORC1 at endolysosomal membranes, the accumulation of the mannose 6-phosphate receptor in and the recruitment of the autophagy marker light chain 3 to enlarged acidic vesicles. We further show that PKD3 depletion strongly inhibited cell spreading and proliferation of TNBC cells, identifying this kinase as a potential novel molecular therapeutic target in TNBC. Together, our data suggest that PKD3 in TNBC cells provides a molecular connection between the Golgi and endolysosomal compartments to enhance proliferative mTORC1-S6K1 signaling.

GIT1 phosphorylation on serine 46 by PKD3 regulates paxillin trafficking and cellular protrusive activity.

The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.

miR149 functions as a tumor suppressor by controlling breast epithelial cell migration and invasion.

Deregulated molecular signaling pathways are responsible for the altered adhesive, migratory, and invasive properties of cancer cells. The different breast cancer subtypes are characterized by the expression of distinct miRNAs, short non-coding RNAs that posttranscriptionally modulate the expression of entire gene networks. Profiling studies have revealed downregulation of miR149 in basal breast cancer. Here, we show that miR149 expression severely impairs cell spreading, migration, and invasion of basal-like breast cancer cells. We identify signaling molecules, including the small GTPases Rap1a and Rap1b, downstream of integrin receptors as miR149 targets, providing an explanation for the defective Src and Rac activation during cell adhesion and spreading upon miR149 expression. Suppression of cell spreading by miR149 could be rescued, at least in part, by expression of constitutively active Rac. Finally, we demonstrate that increased miR149 levels block lung colonization in vivo. On the basis of our findings, we propose that miR149 downregulation in basal breast cancer facilitates the metastatic dissemination of tumor cells by supporting aberrant Rac activation. Cancer Res; 74(18); 5256-65. ©2014 AACR.

An asymmetric interface between the regulatory and core particles of the proteasome.

The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core particle-regulatory particle interface, a site for complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings, one formed by the six ATPase (Rpt) subunits of the regulatory particle and the other by the seven α subunits of the core particle. The Rpt C termini bind to intersubunit cavities in the α-ring, thus directing core particle gating and proteasome assembly. We mapped the Rpt C termini to the α subunit pockets, using a cross-linking approach that revealed an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2-α4, Rpt6-α3 and Rpt3-α2, whereas on the opposite side, the Rpt1, Rpt4 and Rpt5 tails each cross-link to multiple α pockets. Rpt-core particle cross-links are all sensitive to nucleotides, implying that ATP hydrolysis drives dynamic alterations at the core particle-regulatory particle interface.