PTPL1/FAP-1 negatively regulates TRIP6 function in lysophosphatidic acid-induced cell migration

The LIM domain-containing TRIP6 (Thyroid Hormone Receptor-interacting Protein 6) is a focal adhesion molecule known to regulate lysophosphatidic acid (LPA)-induced cell migration through interaction with the LPA2 receptor. LPA stimulation targets TRIP6 to the focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6 at Tyr-55, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration. Here we further demonstrate that a switch from c-Src-mediated phosphorylation to PTPL1/Fas-associated phosphatase-1-dependent dephosphorylation serves as an inhibitory feedback control mechanism of TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This negative regulation requires a direct protein-protein interaction between these two molecules and the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at the sites of adhesions. As a result, LPA-induced association of TRIP6 with Crk and the function of TRIP6 to promote LPA-induced morphological changes and cell migration are inhibited by PTPL1. Together, these results reveal a novel mechanism by which PTPL1 phosphatase plays a counteracting role in regulating TRIP6 function in LPA-induced cell migration.     

TRIP6 enhances lysophosphatidic acid-induced cell migration by interacting with the lysophosphatidic acid 2 receptor

Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA(2) receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA(2) receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130(cas) in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA(2) receptor but not LPA(1) or LPA(3) receptor, indicating a specific role for TRIP6 in regulating LPA(2) receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA(2) receptor and downstream signals involved in cell adhesion and migration.     

RAFTK/Pyk2 mediates LPA-induced PC12 cell migration

The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.     

LPA-induced migration of ovarian cancer cells requires activation of ERM proteins via LPA1 and LPA2

Lysophosphatidic acid (LPA) has been implicated in the pathology of human ovarian cancer. This phospholipid elicits a wide range of cancer cell responses, such as proliferation, trans-differentiation, migration, and invasion, via various G-protein-coupled LPA receptors (LPARs). Here, we explored the cellular signaling pathway via which LPA induces migration of ovarian cancer cells. LPA induced robust phosphorylation of ezrin/radixin/moesin (ERM) proteins, which are membrane-cytoskeleton linkers, in the ovarian cancer cell line OVCAR-3. Among the LPAR subtypes expressed in these cells, LPA₁ and LPA₂, but not LPA₃, induced phosphorylation of ERM proteins at their C-termini. This phosphorylation was dependent on the Gα_12/13/RhoA pathway, but not on the Gα_q/Ca²⁺/PKC or Gα_s/adenylate cyclase/PKA pathway. The activated ERM proteins mediated cytoskeletal reorganization and formation of membrane protrusions in OVCAR-3 cells. Importantly, LPA-induced migration of OVCAR-3 cells was completely abolished not only by gene silencing of LPA₁ or LPA₂, but also by overexpression of a dominant negative ezrin mutant (ezrin-T567A). Taken together, this study demonstrates that the LPA₁/LPA₂/ERM pathway mediates LPA-induced migration of ovarian cancer cells. These findings may provide a potential therapeutic target to prevent metastatic progression of ovarian cancer.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

Matricellular Protein Cyr61 Bridges Lysophosphatidic Acid and Integrin Pathways Leading to Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA₁) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA₁ is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.     

Lysophosphatidic acid induces MDA-MB-231 breast cancer cells migration through activation of PI3K/PAK1/ERK signaling

Background

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.

Methodology/Principal Findings

In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.

Conclusions/Significance

Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.     

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

 Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK^−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130^CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.     

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.     

Crk II silencing down-regulates IGF-IR and inhibits migration and invasion of prostate cancer cells

Crk (C10 regulator of kinase) adaptor proteins are highly expressed in many types of human cancers and often contribute to aggressive cancer phenotypes. Crk II, a member of CRK family, has been reported to regulate cell migration and metastasis in breast cancer cells. However, its role in other cancer types has not been reported. In this study, we investigated the molecular function of Crk II in prostate cancer (PCa) cells (CWR-22rv1) in vitro and using a mouse tumor model. Results showed that Crk II knockdown by shRNA-mediated silencing (Crk II-shRNA) in the PCa cells significantly inhibited both cancer cell migration and invasion in cell culture study. Crk II-shRNA cancer cells also significantly decreased colony formation in vitro, but had no significant reduction of tumor volume after 4 weeks of cancer cell xenografting in vivo when compared to the scramble control. Interestingly, Crk II-shRNA cancer cells showed a greatly reduced level of insulin-like growth factor 1 receptor (IGF-1R) and decreased signaling of the IGF-1R/PI3K/Akt axis upon IGF-1 ligand stimulation. A close interaction between Crk II and IGF-1R was demonstrated upon co-immunoprecipitation of IGF-1R with Crk II protein. Further, treatment of cells with either proteosomal degradation or protein synthesis inhibitor showed higher proportion of ubiquitin-associated IGF-1R and faster degradation of IGF-1R in Crk II-shRNA cells in comparison with that in the control cancer cells. Taken together, these data suggest that Crk II plays an important role in the regulation of IGF-1R protein stability and affects downstream of IGF-1R signaling pathways. Therefore, targeting Crk-II can block IGF-1R growth signaling and suppress cancer cell invasion and progression.     

Lysophosphatidic acid activates TGFBIp expression in human corneal fibroblasts through a TGF-β1-dependent pathway

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease caused by a R124H point mutation in the transforming growth factor-β-induced gene (TGFBI). However, the cellular role of TGFBI and the regulatory mechanisms underlying corneal dystrophy pathogenesis are still poorly understood. Lysophosphatidic acid (LPA) refers to a small bioactive phospholipid mediator produced in various cell types, and binds G protein-coupled receptors to enhance numerous biological responses, including cell growth, inflammation, and differentiation. LPA levels are elevated in injured cornea and LPA is involved in proliferation and wound healing of cornea epithelial cells. Accumulating evidence has indicated a crucial role for LPA-induced expression of TGFBI protein (TGFBIp) through secretion of transforming growth factor-beta1 (TGF-β1). In the current study, we demonstrate that LPA induces TGFBIp expression in corneal fibroblasts derived from normal or GCD2 patients. LPA-induced TGFBIp expression was completely inhibited upon pretreatment with the LPA(1/3) receptor antagonists, VPC32183 and Ki16425, as well as by silencing LPA(1) receptor expression with small hairpin RNA (shRNA) in corneal fibroblasts. LPA induced secretion of TGF-β1 in corneal fibroblasts, and pretreatment with the TGF-β type I receptor kinase inhibitor SB431542 or an anti-TGF-β1 neutralizing antibody also inhibited LPA-induced TGFBIp expression. Furthermore, we show that LPA requires Smad2/3 proteins for the induction of TGFBIp expression. LPA elicited phosphorylation of Smad2/3, and Smad3 specific inhibitor SIS3 or siRNA-mediated depletion of endogenous Smad2/3 abrogates LPA-induced TGFBIp expression. Finally, we demonstrate that LPA-mediated TGFBIp induction requires JNK activation, but not ERK signaling pathways. These results suggest that LPA stimulates TGFBIp expression through JNK-dependent activation of autocrine TGF-β1 signaling pathways and provide important information for understanding the role of phospholipids involved in cornea related diseases.     

Glycoprotein Capture and Quantitative Phosphoproteomics Indicate Coordinated Regulation of Cell Migration upon Lysophosphatidic Acid Stimulation

The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior.The plasma membrane separates the interior of a mammalian cell from the environment. To respond to external signals such as growth factors, cells possess various types of plasma membrane-spanning receptors that communicate to the intracellular signaling machinery in a ligand-regulated manner. G protein-coupled receptors (GPCRs),1 which are integral membrane proteins with seven transmembrane helices, constitute the largest superfamily of cell surface receptors. GPCRs mediate intracellular activation of heterotrimeric G proteins in response to extracellular ligand binding. A plethora of different factors are known to act on GPCRs, including peptide ligands, proteases, nucleotides as well as bioactive lipid molecules such as lysophosphatidic acid (LPA). LPA induces various biological responses including proliferation and migration in a wide range of mammalian cell types and has been implicated in the progression of several human cancers (1, 2). Upon LPA binding to its cognate receptors, heterotrimeric G proteins from the G_i, G_q, and G_12/13 families are activated by guanine nucleotide exchange factors resulting in their dissociation into activated Gα and Gβγ subunits. Activated G protein subunits interact with various effector proteins including phospholipase C and adenylate cyclase isoforms as well as guanine nucleotide exchange factors for Rho family GTPases, which either directly or via second messenger production communicate to cellular kinase signaling. GPCR activation by LPA is also known to trigger the proteolytic activity of ADAM transmembrane metalloproteases, such as ADAM17, which processes epidermal growth factor receptor (EGFR) ligand precursors on the extracellular side to release mature growth factors triggering EGFR activation (3–8). The molecular mechanisms involved in the control of ADAM metalloprotease activity are not clear yet. The resulting transactivation of the EGFR tyrosine kinase provides a link to signaling modules such as mitogen-activated protein kinase cascades and has been implicated in the control of cell proliferation and migration upon LPA treatment (9). Regarding the induction of cell motility upon LPA, previous studies have reported several signaling elements in addition to EGFR transactivation that contribute to this complex cellular behavior. In particular, RhoGTPase-dependent signals that activate downstream effectors such as Rho kinase and focal adhesion kinase are involved in the control of cytoskeletal organization and cell attachment to the surrounding extracellular matrix (ECM) (10, 11). The coordinated regulation of such integrin-mediated interactions is required to enable cell movement and occurs in dedicated macromolecular assemblies such as focal adhesion complexes and hemidesmosomes (12, 13). Despite the key role of integrins, the molecular mechanisms that underlie their functional modulation upon GPCR activation are poorly understood. Moreover, cell-cell contacts such as adherens junctions and desmosomes have to dissociate prior to cell migration. Likewise, it is unclear how the components of these structures, such as members of the cadherin family, might be regulated by GPCR-mediated signaling pathways. Both LPA levels and LPA_1–3 receptor expression are often elevated in cancer patients, and the bioactive lipid acts as a potent inducer of cell migration and invasion in vitro. Due to the key role of protein phosphorylation in LPA-induced signal transmission, comprehensive phosphorylation analysis of regulated proteins might generate new insights into pro-migratory signaling mechanisms in cancer cells. Mass spectrometry (MS)-based analysis has emerged as the key method for unbiased protein phosphorylation studies due to various technological advances in recent years (14, 15). Because of the substoichiometric nature of many site-specific phosphorylation events, phosphopeptides constitute only a small fraction in total peptide samples. Therefore, they need to be efficiently enriched prior to MS analysis, which has become routinely possible by phosphate group-selective purification strategies employing capture reagents such as immobilized metal ion affinity chromatography or titanium dioxide beads (16). Moreover, phosphopeptide analysis has benefited enormously from the availability of hybrid mass spectrometers that combine the sensitivity and speed of linear ion traps with the high resolution and accuracy of orbitrap mass analyzers (17). These advances together with quantitative approaches such as stable isotope labeling by amino acids in cell cultures (SILAC) (18, 19) and substantial progress in computational proteomics (20) now allow for concomitant identification and quantification of several thousand phosphorylation sites from single cellular extracts (21–23).We previously analyzed cell signaling responses in A498 kidney carcinoma cells upon LPA and heparin-binding EGF-like growth factor treatment by monitoring phosphorylation changes in total cell lysate and protein kinase-enriched fractions (24). In a complementary approach, we now aimed for a systematic survey of LPA-induced phosphorylation changes on plasma membrane proteins and their interaction partners. Furthermore, we were interested in time-resolved analysis of LPA-induced phosphorylation changes on ADAM17 and the EGFR to gain further insights into possible mechanisms underlying the still enigmatic EGFR transactivation process. In our present study, we therefore analyzed SCC-9 squamous carcinoma cells due to their pronounced EGFR transactivation response upon LPA. As plasma membrane proteins usually contain covalently attached carbohydrate structures, we performed lectin affinity enrichment of glycosylated proteins prior to SILAC-based quantitative phosphoproteomics (25). This experimental strategy enabled us to acquire in-depth data about LPA regulation of diverse glycoproteins and revealed coordinated phosphoregulation of cell adhesion proteins as likely mechanism underlying cell migratory behavior.     

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Abstract

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.