Veerle De Corte

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3ζ

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3ζ as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.

Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent pro tein (GFP)‐tagged gelsolin in MDCK‐AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I. In organ culture assays, MDCK cells expressing gelsolin–GFP invaded pre‐cultured chick heart fragments. Gelsolin expression inhibited E‐cadherin‐mediated cell aggregation but did not disrupt the E‐cadherin–catenin complex. Co‐expression of dominant‐negative Rac1N17, but not RhoAN19 or Cdc42N17, counteracted gelsolin‐induced invasion, suggesting a requirement for Rac1 activity. Increased ARF6, PLD or PIP5K 1α activity canceled out gelsolin‐induced invasion. Furthermore, we found that invasion induced by gelsolin is dependent on Ras activity, acting through the PI3K–Rac pathway via the Ras guanine nucleotide exchange factor Sos‐1. These findings establish a connection between gelsolin and the Ras oncogenic signaling pathway.

Phosphatidylinositol 4,5-bisphosphate specifically stimulates PP60c-src catalyzed phosphorylation of gelsolin and related actin-binding proteins

Gelsolin is a widely distributed Ca2+‐dependent regulator of the cortical actin network. We demonstrate that gelsolin is phosphorylated by pp60c‐src and that this phosphorylation is dramatically enhanced by phosphatidylinositol 4,5‐bisphosphate (PIP2), known to specifically interact with gelsolin. Other phospholipids display only a marginal effect. pp56lck, a tyrosine kinase of the same family, does not phosphorylate gelsolin. Other mammalian actin‐binding proteins such as profilin and CapG but also fragmin from Physarum polycephalum are similar targets for PIP2‐stimulated pp60c‐src phosphorylation.

Nuclear Actin-Binding Proteins as Modulators of Gene Transcription

Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin‐binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II‐containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain regulates its intracellular distribution and its interaction with F-actin in transfected cells and in in vitro assays. Phosphorylated wild-type L-plastin localised to the actin cytoskeleton in transfected Vero cells. Ser5Ala substitution reduced the capacity of L-plastin to localise with peripheral actin-rich membrane protrusions. Conversely, a Ser5Glu variant mimicking a constitutively phosphorylated state, accumulated in actin-rich regions and promoted the formation of F-actin microspikes in two cell lines. Similar to phosphorylated wild-type L-plastin, this variant remained associated with cellular F-actin in detergent-treated cells, whereas the Ser5Ala variant was almost completely extracted. When compared with non-phosphorylated protein, phosphorylated L-plastin and the Ser5Glu variant bound F-actin more efficiently in an in vitro assay. Importantly, expression of L-plastin elicited collagen invasion in HEK293T cells, in a manner dependent on Ser5 phosphorylation. Based on our findings, we propose that conversely to other calponin homology (CH)-domain family members, phosphorylation of L-plastin switches the protein from a low-activity to a high-activity state. Phosphorylated L-plastin might act as an integrator of signals controlling the assembly of the actin cytoskeleton and cell motility in a 3D-space.

Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin β but not importin α. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.

The Nucleo-cytoplasmic Actin-binding Protein CapG Lacks a Nuclear Export Sequence Present in Structurally Related Proteins

Despite thorough structure-function analyses, it remains unclear how CapG, a ubiquitous F-actin barbed end capping protein that controls actin microfilament turnover in cells, is able to reside in the nucleus and cytoplasm, whereas structurally related actin-binding proteins are predominantly cytoplasmic. Here we report the molecular basis for the different subcellular localization of CapG, severin, and fragminP. Green fluorescent protein-tagged fragminP and severin accumulate in the nucleus upon treatment of transfected cells with the CRM1 inhibitor leptomycin B. We identified a nuclear export sequence in severin and fragminP, which is absent in CapG. Deletion of amino acids Met1–Leu27resulted in nuclear accumulation of severin and fragminP. Tagging this sequence to CapG triggered nuclear export, whereas mutation of single leucine residues (Leu17, Leu21, and Leu27) in the export sequence inhibited nuclear export. Based on these findings, a nuclear export signal was identified in myopodin, a muscle-specific actin-binding protein, and the Bloom syndrome protein, a RecQ-like helicase. Deletion of the myopodin nuclear export sequence blocked invasion into collagen type I of C2C12 cells transiently overexpressing myopodin. Our findings explain regulated subcellular targeting of distinct classes of actin-binding proteins.

A New Role for Nuclear Transport Factor 2 and Ran: Nuclear Import of CapG

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin‐permeabilized cells, neither GTPγS nor the GTP hydrolysis‐deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2–Ran complex. NTF2–Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.

β‐casein‐derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT‐8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility‐promoting factor, identified as a 13mer β‐casein‐derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin‐like metalloprotease, and a collagen‐associated trypsin‐like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a β‐casein source. The pro‐ invasive 13mer peptide‐signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21‐ activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3‐kinase. Delta opioid receptor (δOR) is a candidate receptor for the 13mer peptide since naloxone, an δOR antagonist, blocks both δOR serine phosphorylation and 13mer peptide‐mediated invasion.

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein‐CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61‐ECFP‐CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.