Characterization of the FAM110 gene family

We have previously characterized the centrosome/spindle pole-associated protein (CSPP) involved in cell cycle progression. The open reading frame C20orf55 was identified in a yeast two-hybrid screen in a search for CSPP-interacting proteins. A homology search revealed that C20orf55 belongs to a gene family consisting of three members that have not yet been described. The HUGO Nomenclature Committee has assigned these genes the names FAM110A-FAM110C. Studies of transfectants showed that the FAM110 proteins localized to centrosomes and accumulated at the microtubule organization center in interphase and at spindle poles in mitosis. In addition, overexpression of FAM110C induced microtubule aberrancies. Our data also indicated a cell cycle-regulated expression of FAM110A. Moreover, ectopic expression of FAM110B and FAM110C proteins impaired cell cycle progression in G1 phase. To summarize, we have characterized a novel family of genes encoding proteins with distinct conserved motifs, of which all members localize to centrosomes and spindle poles.     

DRG2 knockdown induces Golgi fragmentation via GSK3β phosphorylation and microtubule stabilization

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3β (GSK3β)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3β. Tau, a target of GSK3β, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3β phosphorylation, and microtubule stability.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

Overcoming Amino-Nogo-induced Inhibition of Cell Spreading and Neurite Outgrowth by 12-O-Tetradecanoylphorbol-13-acetate-type Tumor Promoters

The N-terminal domain of NogoA, called amino-Nogo, inhibits axonal outgrowth and cell spreading via a largely unknown mechanism. In the present study, we show that amino-Nogo decreases Rac1 activity and inhibits fibroblast spreading. 12-O-Tetradecanoylphorbol-13-acetate-type tumor promoters, such as phorbol 12-myristate 13-acetate (PMA) and teleocidin, increase Rac1 activity and overcome the amino-Nogo-induced inhibition of cell spreading. The stimulating effect of tumor promoters on cell spreading requires activation of protein kinase D and the subsequent activation of Akt1. Furthermore, we identified Akt1 as a new signaling component of the amino-Nogo pathway. Akt1 phosphorylation is decreased by amino-Nogo. Activation of Akt1 with a cell-permeable peptide, TAT-TCL1, blocks the amino-Nogo inhibition. Finally, we provide evidence that these signaling pathways operate in neurons in addition to fibroblasts. Our results suggest that activation of protein kinase D and Akt1 are approaches to promote axonal regeneration after injury.     

Interaction of N-WASP with hnRNPK and its role in filopodia formation and cell spreading

N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center.     

Contra-regulation of calcium- and integrin-binding protein 1-induced cell migration on fibronectin by PAK1 and MAP kinase signaling

Calcium- and integrin-binding protein 1 (CIB1) has been shown to be involved in cell spreading and migration. The signaling events regulated by CIB1 during cell migration are poorly understood. Here we found that accumulation of CIB1 at the tip of the filopodia requires an intact cytoskeleton. Depletion of CIB1 using shRNA affects formation of FAK- and phosphotyrosine-rich focal adhesions without affecting stress fiber formation. Overexpression of CIB1 results in cell migration on fibronectin and Erk1/2 MAP kinase activation. CIB1-induced cell migration is dependent upon Erk1/2 activation, since it is inhibited by the MEK-specific inhibitor PD98059. Furthermore, CIB1-induced cell migration, as well as Erk1/2 activation, is dependent on PKC, Src family kinases as well as PI-3 kinase as it is inhibited by bisindolylmaleimide 1, PP2, and wortmannin, respectively, in a dose-dependent manner. Co-expression of dominant-negative Cdc42 completely abolished CIB1-induced cell migration. Additionally, co-expression of constitutively active, but not dominant negative PAK1, a CIB1 binding protein, inhibited CIB1-induced cell migration. These results suggest that CIB1 positively regulates cell migration and is necessary for the recruitment of FAK to the focal adhesions. Furthermore, CIB1-induced cell migration is dependent on MAP kinase signaling and its function is attenuated by PAK1.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Focal Adhesion Kinase Functions as an Akt Downstream Target in Migration of Colorectal Cancer Cells

Migration is a complex process that, besides its various physiological functions in embryogenesis and adult tissues, plays a crucial role in cancer cell invasion and metastasis. The focus of this study is the involvement and collaboration of Akt, focal adhesion kinase (FAK), and Src kinases in migration and invasiveness of colorectal cancer cells. We show that all three kinases can be found in one protein complex; nevertheless, the interaction between Akt and Src is indirect and mediated by FAK. Interestingly, induced Akt signaling causes an increase in tyrosine phosphorylation of FAK, but this increase is attenuated by the Src inhibitor SU6656. We also show that active Akt strongly stimulates cell migration, but this phenomenon is fully blocked by FAK knockdown or partly by inhibition of Src kinase. In addition, we found that all three kinases were indispensable for the successful invasion of colorectal cancer cells. Altogether, the presented data bring new insights into the mechanism how the phosphatidylinositol-3-kinase (PI3-K)/Akt pathway can influence migration of colorectal adenocarcinoma cells. Because FAK is indispensable for cell movements and functions downstream of Akt, our results imply FAK kinase as a potential key molecule during progression of tumors with active PI3-K/Akt signaling.     

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.     

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.     

PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane–substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5–10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells.We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

CD44-mediated elongated T cell spreading requires Pyk2 activation by Src family kinases, extracellular calcium, phospholipase C and phosphatidylinositol-3 kinase

The proline-rich tyrosine kinase 2, Pyk2, is a focal adhesion related kinase expressed in T cells that is tyrosine phosphorylated and activated by integrin, chemokine or T cell receptor stimulation. Ligation of the cell adhesion molecule CD44 also induces Pyk2 phosphorylation and T cell spreading, and this is negatively regulated by the protein tyrosine phosphatase CD45. Here, we identify the activation requirements for Pyk2 and demonstrate its requirement for CD44-mediated elongated T cell spreading. Upon CD44-mediated cell spreading, Pyk2 was recruited to CD44 clusters in both CD45⁺ and CD45⁻ T cells, yet was more strongly phosphorylated in T cells lacking CD45. In these cells, Pyk2 phosphorylation was dependent on Src family kinase activity and required actin polymerisation, phosphatidylinositol-3 kinase and phospholipase C activity as well as extracellular calcium. Inhibition of any of these events prevented Pyk2 phosphorylation and T cell spreading. Transfection of a truncated form of Pyk2 lacking the kinase domain, PRNK, inhibited CD44-mediated cell spreading, demonstrating an important role for Pyk2. However, inhibition of microtubule turnover by Taxol prevented elongated T cell spreading but did not affect Pyk2 phosphorylation, indicating that microtubule reorganisation is downstream, or independent, of Pyk2 phosphorylation. Together this demonstrates that multiple factors are required for CD44-induced Pyk2 activation, which plays a critical role in CD44-mediated elongated T cell spreading.     

Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of alpha v beta 3 and alpha 5 beta 1 integrins

Protein kinase B (PKB)/Akt is known to promote cell migration, and this may contribute to the enhanced invasiveness of malignant cells. To elucidate potential mechanisms by which PKB/Akt promotes the migration phenotype, we have investigated its role in the endosomal transport and recycling of integrins. Whereas the internalization of alpha v beta 3 and alpha 5 beta 1 integrins and their transport to the recycling compartment were independent of PKB/Akt, the return of these integrins (but not internalized transferrin) to the plasma membrane was regulated by phosphatidylinositol 3-kinases and PKB/Akt. The blockade of integrin recycling and cell spreading on integrin ligands effected by inhibition of PKB/Akt was reversed by inhibition of glycogen synthase kinase 3 (GSK-3). Moreover, expression of nonphosphorylatable active GSK-3 beta mutant GSK-3 beta-A9 suppressed recycling of alpha 5 beta 1 and alpha v beta 3 and reduced cell spreading on ligands for these integrins, indicating that PKB/Akt promotes integrin recycling by phosphorylating and inactivating GSK-3. We propose that the ability of PKB/Akt to act via GSK-3 to promote the recycling of matrix receptors represents a key mechanism whereby integrin function and cell migration can be regulated by growth factors.     

Cryptotanshinone inhibits macrophage migration by impeding F-actin polymerization and filopodia extension

We evaluated the anti-inflammatory effects of cryptotanshinone and tanshinone IIA, two major tanshinones isolated from Salvia miltiorrhiza, on chemoattractant-induced cell migration in RAW264.7 macrophages. Results showed that cryptotanshinone inhibited cell migration toward complement 5a (C5a) and macrophage inflammatory protein-1alpha (MIP-1alpha) in a concentration-dependent manner. In contrast, tanshinone IIA displayed less or even no effect on cell migration evoked by these chemoattractants. Both C5a- and MIP-1alpha-induced migration were clearly inhibited by cytochalasin B (an inhibitor of actin polymerization), but not by colchicine (an inhibitor of microtubule polymerization). Fluorescence staining demonstrated that cryptotanshinone as well as cytochalasin B, effectively reversed cell polarization and filopodia extension induced by both chemoattractants. Furthermore, C5a-evoked increase in F-actin fluorescence intensity was significantly suppressed by cryptotanshinone. Based on these observations, we suggest that cryptotanshinone exerts anti-migrating activity possibly by impeding F-actin polymerization and filopodia formation.     

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.     

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.     

Differential phosphorylation of Akt1 and Akt2 by protein kinase CK2 may account for isoform specific functions

Akt (also known as PKB) is a survival kinase frequently up-regulated in cancer; three isoforms of Akt exist, and among them Akt1 and Akt2 are the most widely and highly expressed. They share the same structure and activation mechanism and have many overlapping functions; nevertheless isoform-specific roles and substrates have been reported, which are expected to rely on sequence diversities. In particular, a special role in differentiating Akt1 and Akt2 isoforms has been assigned to the linker region, a short segment between the PH and the catalytic domains. We have previously found that a residue in the linker region (Ser129) is directly phosphorylated by protein kinase CK2 in Akt1; the phosphorylation of the homologous residue in Akt2 (Ser131) has never been analyzed. Here we show that Akt2, endogenously or ectopically expressed in different cell lines, is not phosphorylated on Ser131 by CK2, while in vitro recombinant Akt2 is a CK2 substrate. These data support the hypothesis that in vivo a steric hindrance occurs which prevents the access to the CK2 site. Additionally, we have found that Ser129 phosphorylation is involved in the recognition of the Akt1-specific substrate palladin; this observation provides an explanation of why Akt2, lacking Ser131 phosphorylation in the linker region, has a low efficiency in targeting palladin. CK2-dependent phosphorylation is therefore a crucial event which, discriminating between Akt1 and Akt2, can account for different substrate specificities, and, more in general, for fine tuning of Akt activity in the control of isoform-dependent processes.