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Invadopodia formation and extracellular matrix degradation are key events during cancer cell invasion, yet little is known about mechanisms mediating these processes. Here, we report that Rab40b plays a key role in mediating invadopodia function during breast cancer cell invasion. We also identify Tks5 (also known as SH3PXD2A), a known Src kinase substrate, as a new Rab40b effector protein and show that Tks5 functions as a tether that mediates Rab40b-dependent targeting of transport vesicles containing MMP2 and MMP9 to the extending invadopodia. Importantly, we also demonstrate that Rab40b and Tks5 levels are regulated by known tumor suppressor microRNA miR-204. This is the first study that identifies a new Rab40b-Tks5- and miR-204-dependent invadopodia transport pathway that regulates MMP2 and MMP9 secretion, and extracellular matrix remodeling during cancer progression., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Endocytosis, which is a key process in eukaryotic cells, has a central role in maintaining cellular homeostasis, nutrient uptake, development and downregulation of signal transduction. This complex process depends on several protein-protein interactions mediated by specific modules. One such module is the EH domain. The EH-domain-containing proteins comprise a family that includes four vertebrate members (EHD1-EHD4) and one Drosophila ortholog, Past1. We used Drosophila as a model to understand the physiological role of this family of proteins. We observed that the two predicted Past1 transcripts are differentially expressed both temporally and spatially during the life cycle of the fly. Endogenous Past1 as well as Past1A and Past1B, expressed from plasmids, were localized mainly to the membrane of Drosophila-derived cells. We generated mutants in the Past1 gene by excising a P-element inserted in it. The Past1 mutants reached adulthood but died precociously. They were temperature sensitive and infertile because of lesions in the reproductive system. Garland cells that originated from Past1 mutants exhibited a marked decrease in their ability to endocytose fluorescently labeled avidin. Genetic interaction was found between Past1 and members of the Notch signaling pathway, suggesting a role for Past1 in this developmentally crucial signaling pathway.

Clathrin-coated pits assemble on the plasma membrane to select and sequester proteins within coated vesicles for delivery to intracellular compartments. Although a host of clathrin-associated proteins have been identified, much less is known regarding the interactions between clathrin-associated proteins or how individual proteins influence the function of other proteins. In this study, we present evidence of a functional relationship between two clathrin-associated proteins in Dictyostelium, Hip1r and epsin. Hip1r-null cells form fruiting bodies that yield defective spores that lack the organized fibrils typical of wild-type spores. This spore coat defect leads to formation of round, rather than ovoid, spores in Hip1r-null cells that exhibit decreased viability. Like Hip1r-null cells, epsin-null cells also construct fruiting bodies with round spores, but these spores are more environmentally robust. Double-null cells that harbor deletions in both epsin and Hip1r form fruiting bodies, with spores identical in shape and viability to Hip1r single-null cells. In the growing amoeba, Hip1r is phosphorylated and localizes to puncta on the plasma membrane that also contain epsin. Both the phosphorylation state and localization of Hip1r into membrane puncta require epsin. Moreover, expression of the N-terminal ENTH domain of epsin is sufficient to restore both the phosphorylation and the restricted localization of Hip1r within plasma membrane puncta. The results from this study reveal a novel interaction between two clathrin-associated proteins during cellular events in both growing and developing Dictyostelium cells.

Cargo selectivity is a hallmark of clathrin-mediated endocytosis. A wide range of structurally unrelated internalization signals specify the preferential clustering of transmembrane cargo into clathrin coats forming on the plasma membrane. Intriguingly, the classical endocytic adaptor AP-2 appears to recognize only a subset of these endocytic sorting signals. New data now reveal the molecular basis for recognition of other internalization signals, including post-translationally appended ubiquitin, by clathrin-coat-associated sorting proteins (CLASPs). Curiously, structurally related ubiquitin-recognition modules are shared by select CLASPs and the 26S proteasome, and recent work indicates that both display similar requirements for ubiquitin binding. During endocytosis, these modules engage oligoubiquitylated cargo in the form of polyubiquitin chains and/or multiple single ubiquitin molecules appended to different acceptor lysines. Functional separation between clathrin-mediated endocytosis and proteasome-dependent proteolysis is probably ensured by temporally regulated, local assembly of ubiquitin-tagged membrane cargo at sorting stations on the cell surface, shielding ubiquitin sorting signals from the proteasome. Thus, an expanded repertoire of CLASPs couples the process of clathrin-coat assembly with high-fidelity incorporation of assorted, cargo-specific sorting signals.

Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25alpha, which are required for the assembly of MO25alpha into a complex with STRADalpha and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADalpha-MO25alpha, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADalpha, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.