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PubMed2007

Statistical validation of the root-mean-square-distance, a measure of protein structural proximity.

Despite its well-documented limitations, the root-mean-square-distance (rmsd) between pairs of equivalent atoms is routinely used to monitor the degree of similarity between two optimally superposed protein three-dimensional structures. A robust method for assessing the statistical significance of the difference between two rmsd values is presented here. It is based on the comparison of two protein structures through the correlation coefficient between equivalent inter-atomic distances and the subsequent application of the Fisher transformation that allows one to estimate the probability of identity between two correlation coefficient values. The relationship between the rmsd and Fisher correlation coefficient allows then to estimate the statistical significance of the difference between two rmsd values. Such a procedure is exemplified with the analysis of the possible classifications of the immunoglobulin-like domains of filamin and is compared to related estimations of structural similarity. The possibility to estimate the probability of the difference between two rmsd values can be used to optimize the protein structural classifications and comparisons, independent of the procedure used to derive the rmsds.

Algorithms↗
PubMed2006

Stress-dependent elasticity of composite actin networks as a model for cell behavior.

Networks of filamentous actin cross-linked with the actin-binding protein filamin A exhibit remarkable strain stiffening leading to an increase in differential elastic modulus by several orders of magnitude over the linear value. The variation of the frequency dependence of the differential elastic and loss moduli as a function of prestress is consistent with that observed in living cells, suggesting that cell elasticity is always measured in the nonlinear regime, and that prestress is an essential control parameter.

Actins↗
PubMed2004

Crystallization and phasing of focal adhesion protein 52 from Gallus gallus.

Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal alpha-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1 A resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9 A wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit.

Animals↗
PubMed2006

Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction.

The transmembrane peptidase prostate-specific membrane antigen (PSMA) is universally upregulated in the vasculature of solid tumors, but its functional role in tumor angiogenesis has not been investigated. Here we show that angiogenesis is severely impaired in PSMA-null animals and that this angiogenic defect occurs at the level of endothelial cell invasion through the extracellular matrix barrier. Because proteolytic degradation of the extracellular matrix is a critical component of endothelial invasion in angiogenesis, it is logical to assume that PSMA participates in matrix degradation. However, we demonstrate a novel and more complex role for PSMA in angiogenesis, where it is a principal component of a regulatory loop that is tightly modulating laminin-specific integrin signaling and GTPase-dependent, p21-activated kinase 1 (PAK-1) activity. We show that PSMA inhibition, knockdown, or deficiency decreases endothelial cell invasion in vitro via integrin and PAK, thus abrogating angiogenesis. Interestingly, the neutralization of beta(1) or the inactivation of PAK increases PSMA activity, suggesting that they negatively regulate PSMA. This negative regulation is mediated by the cytoskeleton as the disruption of interactions between the PSMA cytoplasmic tail and the anchor protein filamin A decreases PSMA activity, integrin function, and PAK activation. Finally, the inhibition of PAK activation enhances the PSMA/filamin A interaction and, thus, boosts PSMA activity. These data imply that PSMA participates in an autoregulatory loop, wherein active PSMA facilitates integrin signaling and PAK activation, leading to both productive invasion and downregulation of integrin beta(1) signaling via reduced PSMA activity. Therefore, we have identified a novel role for PSMA as a true molecular interface, integrating both extracellular and intracellular signals during angiogenesis.

Animals↗
PubMed2006

Loss of FilaminC (FLNc) results in severe defects in myogenesis and myotube structure.

FilaminC (FLNc) is the muscle-specific member of a family of actin binding proteins. Although it interacts with many proteins involved in muscular dystrophies, its unique role in muscle is poorly understood. To address this, two models were developed. First, FLNc expression was stably reduced in C2C12 myoblasts by RNA interference. While these cells start differentiation normally, they display defects in differentiation and fusion ability and ultimately form multinucleated "myoballs" rather than maintain elongated morphology. Second, a mouse model carrying a deletion of last 8 exons of Flnc was developed. FLNc-deficient mice die shortly after birth, due to respiratory failure, and have severely reduced birth weights, with fewer muscle fibers and primary myotubes, indicating defects in primary myogenesis. They exhibit variation in fiber size, fibers with centrally located nuclei, and some rounded fibers resembling the in vitro phenotype. The similarity of the phenotype of FLNc-deficient mice to the filamin-interacting TRIO null mice was further confirmed by comparing FLNc-deficient C2C12 cells to TRIO-deficient cells. These data provide the first evidence that FLNc has a crucial role in muscle development and maintenance of muscle structural integrity and suggest the presence of a TRIO-FLNc-dependent pathway in maintaining proper myotube structure.

Animals↗
PubMed2001

Disruption of the mouse mu-calpain gene reveals an essential role in platelet function.

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu-calpain-deficient mice are viable and fertile. The complete deficiency of mu-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alphaIIbbeta3 integrin, talin, and ABP-280 (filamin). However, mu-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alphaIIbbeta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.

Adenosine Diphosphate↗
PubMed2003

Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components.

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Actins↗
PubMed2005

The association of caveolae, actin, and the dystrophin-glycoprotein complex: a role in smooth muscle phenotype and function?

Smooth muscle cells exhibit phenotypic and mechanical plasticity. During maturation, signalling pathways controlling actin dynamics modulate contractile apparatus-associated gene transcription and contractile apparatus remodelling resulting from length change. Differentiated myocytes accumulate abundant caveolae that evolve from the structural association of lipid rafts with caveolin-1, a protein with domains that confer unique functional properties. Caveolae and caveolin-1 modulate and participate in receptor-mediated signalling, and thus contribute to functional diversity of phenotypically similar myocytes. In mature smooth muscle, caveolae are partitioned into discrete linear domains aligned with structural proteins that tether actin to the extracellular matrix. Caveolin-1 binds with beta-dystroglycan, a subunit of the dystrophin glycoprotein complex (DGC), and with filamin, an actin binding protein that organizes cortical actin, to which integrins and focal adhesion complexes are anchored. The DGC is linked to the actin cytoskeleton by a dystrophin subunit and is a receptor for extracellular laminin. Thus, caveolae and caveolin-associated signalling proteins and receptors are linked via structural proteins to a dynamic filamentous actin network. Despite development of transgenic models to investigate caveolins and membrane-associated actin-linking proteins in skeletal and cardiac muscle function, only superficial understanding of this association in smooth muscle phenotype and function has emerged.

Actins↗
PubMed1991

Actin filaments regulate epithelial Na+ channel activity.

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

Actins↗
PubMed1995

cAMP-independent regulation of CFTR by the actin cytoskeleton.

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.

Actins↗
PubMed1990

Isoform distribution and tissue contents of contractile and cytoskeletal proteins in hypertrophied smooth muscle from rat portal vein.

Growth of the smooth muscle in the rat portal vein was initiated by an increased transmural pressure. After 7 days, the cross-sectional area of the vessel wall and the maximal active force of the longitudinal muscle layer had increased twofold. Electron microscopy showed that the cell cross-sectional area was increased, suggesting cellular hypertrophy. Increased amounts of intermediate (10 nm) filaments were observed in the hypertrophied cells. The hypertrophied vessels had decreased DNA content per unit wet weight compared with the control vessels (hypertrophied, 1.5 +/- 0.1; control, 1.9 +/- 0.1 micrograms/mg; p less than 0.01). Protein composition was studied with electrophoretic methods. Compared with control preparations the hypertrophied veins had similar myosin and actin contents per unit wet weight (myosin: hypertrophied, 4.4 +/- 0.8; control, 5.9 +/- 0.9; actin: hypertrophied 12.2 +/- 0.6; control, 11.8 +/- 1.0 mg/g). Two different forms of the myosin heavy chain were detected with 5% sodium dodecyl sulfate-polyacrylamide gels. The proportion of the lower molecular weight heavy chain relative to total heavy chain content was about 30% and similar in both preparations. The relation filamin/myosin was increased in the hypertrophied vessels. Pyrophosphate gel electrophoresis revealed two protein bands, with an increase in the slower migrating band in the hypertrophied vessels possibly reflecting an increase in filamin content in the extracts. In the control portal vein alpha-actin is the dominating isoform constituting about 55% of total actin. In hypertrophied vessels, alpha-actin decreased (by 15%) and gamma-actin increased (by 20%). The portal vein contained desmin and vimentin in a ratio of about 6:1. The hypertrophied vessels showed a marked increase in the amount of these proteins (desmin/actin: hypertrophied, 0.32; control, 0.14). In conclusion, during pressure-induced growth of the portal vein, contractile protein contents increase in proportion to the increase in weight. A change in isoforms of actin occurs but no evidence for a change in myosin isoforms was found. The structural proteins increase relative to tissue weight, possibly associated with the increased number of intermediate filaments demonstrated with electron microscopy.

Actins↗
PubMed2006

Beta1 and beta3 integrins cooperate to induce syndecan-4-containing cross-linked actin networks in human trabecular meshwork cells.

PURPOSE: To characterize the molecular composition of cross-linked actin networks (CLANs) and the regulation of their formation by integrins in normal human trabecular meshwork (TM) cells. CLANs have been observed in steroid-treated and glaucomatous TM cells and have been suggested to contribute to decreased outflow facility by altering the contractility of the TM. METHODS: Immunofluorescence microscopy was used to identify molecular components of CLANs and quantitate CLAN formation in HTM cells plated on coverslips coated with various extracellular matrix (ECM) proteins (fibronectin, types I and IV collagen, and vitronectin), vascular cell adhesion molecule (VCAM)-1, or activating antibodies against beta1, beta3, or alpha2beta1 integrins. These integrin antibodies were also used as soluble ligands. RESULTS: CLAN vertices contained the actin-binding proteins alpha-actinin and filamin and the signaling molecules syndecan-4 and PIP2. CLANs lacked Arp3 and cortactin. CLAN formation was dependent on the ECM substrate and was significantly higher on fibronectin and VCAM-1 compared with vitronectin, types I or IV collagen. Adsorbed beta1 integrin antibodies also induced CLANs, whereas adsorbed beta3 or alpha2beta1 integrin antibodies did not. Soluble beta3 integrin antibodies, however, induced CLANs and actually enhanced CLAN formation in cells spread on fibronectin, VCAM-1, type I or type IV collagen, or beta1 integrin antibodies. CONCLUSIONS: CLANs are unique actin-branched networks whose formation can be regulated by beta1 and beta3 integrin signaling pathways. Thus, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and hence could participate in regulating cytoskeletal events previously demonstrated to be involved in controlling outflow facility.

Actin Cytoskeleton↗
PubMed2002

Periventricular nodular heterotopia: report of a pediatric series.

Periventricular nodular heterotopia is a malformation that occurs in both males and females and is associated with a variety of clinical and neuroradiologic signs. A gene called filamin-1 (FLN-1) has recently been identified. We review the clinical and imaging findings from a series of pediatric patients with periventricular nodular heterotopia. Five patients (three males and two females; age range = 4-18 years) were investigated. In our series, periventricular nodular heterotopia can be the common denominator in different conditions. Periventricular nodular heterotopia can occur alone or be associated with cortical malformations. Epilepsy was present in three of the five patients and was resistant to drugs in one female. Mental retardation was present in three of the five patients. Two male patients had normal intelligence, with no cortical anomalies; patient 3 had unilateral periventricular nodular heterotopia. The associated malformations were more severe in the female patients and slight only in patient 1. The two females showed anomalies rarely reported in association with bilateral periventricular nodular heterotopia. We believe that other genes can be involved in children with atypical neuroradiologic periventricular nodular heterotopia. No mutations were detected in 6 of the 48 exons of the FLN-1 gene, although this does not allow any definitive conclusions to be reached. We conclude that our series of patients with periventricular nodular heterotopia clearly highlights the complexity of the clinical, neurologic, and neuroradiologic characteristics associated with this malformation.

Abnormalities, Multiple↗
PubMed2002

Familial periventricular heterotopia: missense and distal truncating mutations of the FLN1 gene.

OBJECTIVE: To examine the clinical and MRI associations in bilateral periventricular nodular heterotopia (BPNH) (MIM # 300049) in two families segregating a missense mutation and a C-terminal deletion of the filamin 1(FLN1) gene. BACKGROUND: Classical familial BPNH, an X-linked dominant disorder, has been associated with protein truncations or splicing mutations, which tend to cluster at the N-terminal of the FLN1 protein, causing severe predicted loss of the protein function. The clinical syndrome includes symmetrical contiguous nodular heterotopia lining the lateral ventricles, epilepsy, mild retardation to normal cognitive level in affected females, and prenatal lethality in hemizygous boys. METHODS: Clinical examination, cognitive testing, MRI, mutation analysis (direct sequencing, single-strand conformation polymorphism) in seven patients from two families with BPNH. RESULTS: In Family 1, harboring an A > T change in exon 2 (E82V), heterotopic nodules were few, asymmetric, and noncontiguous. Five boys born from affected females had died unexpectedly early in life. In Family 2, harboring an 8 base pair deletion in exon 47 (7627_7634del TGTGCCCC), heterotopic nodules were thick and contiguous. Affected females in both families showed normal to borderline IQ and epilepsy. CONCLUSION: Missense mutations and distal truncations consistent with partial loss of FLN1 function cause familial BPNH with the classical clinical phenotype including epilepsy and mild mental retardation, if any. However, missense mutations have milder anatomic consequences in affected females and are possibly compatible with live birth but short survival of boys.

Abortion, Spontaneous↗
PubMed1993

Cytoarchitecture of size-excluding compartments in living cells.

By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from otherwise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent non-excluding domains. The height of the distal excluding compartments was generally < or = 50% of the height in the adjacent non-excluding compartment. An electron-dense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin nonmuscle filamin (ABP280) and alpha-actinin. F-actin and ABP280, but not alpha-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the trans-Golgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.

3T3 Cells↗
PubMed1995

A high molecular mass protein isolated from chicken gizzard: its localization at the dense plaques and dense bodies of smooth muscle and the Z-disks of skeletal muscle.

We purified a 450 kDa protein from a low-salt alkaline extract of chicken gizzard smooth muscle. This high molecular mass protein could be extracted with the low-salt alkaline solution at 37 degrees C but not at 4 degrees C. The 450 kDa protein was isolated from the extract by ammonium sulfate fractionation and following sequential column chromatography using hydroxylapatite, DEAE-Cellulofine A-800m and phenyl-Sepharose CL-4B resins. The partially purified protein molecule resembled a flexible rod with a globular head and an irregular-shaped tail. Its length was approximately 300 nm. The nucleotide sequence of the partial cDNA encoding this protein was determined and analyzed with a data base. The analysis showed that the protein revealed significant homology with the rod region of chicken filamin (57% homology in amino acid sequence). Immunoblot analysis showed that an affinity-purified antibody reacted exclusively with the 450 kDa protein band of smooth, skeletal and cardiac muscle tissues. By indirect immunofluorescence microscopy, we examined the localization of the 450 kDa protein in smooth and skeletal muscle cells. The affinity-purified antibody against the 450 kDa protein stained the dense plaques and dense bodies of smooth muscle, the peripheral region of Z-disks and the subsarcolemmal region of skeletal muscle. Immunoelectron microscopy confirmed the localization of the 450 kDa protein at the peripheral regions of the actin anchoring structures mentioned above. Judging from its amino acid sequence, molecular size, molecular shape, immunological reactivity and localization in muscle cells, the 450 kDa protein seemed to be a new component associated with the actin-anchoring structures of muscle tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Observations of microfilament bundles in living cells microinjected with fluorescently labelled contractile proteins.

Fluorescently labelled contractile proteins (alpha-actinin and filamin) were used to study the dynamic nature of three types of microfilament bundles: myofibrils, stress fibres and polygonal networks. Cultured muscle and non-muscle cells that were microinjected with fluorescent alpha-actinin rapidly incorporated the labelled protein into Z-bands, stress fibre densities and the polygonal foci. Living, injected cells were then observed for varying periods of time, and changes in orientation and periodicity of the myofibrils, stress fibres and polygonal networks were recorded. Permeabilized cells were also reacted with fluorescently labelled proteins and with contractile protein antibodies in order to analyse further the changes taking place in the myofibrils and stress fibres. In both living cardiac myocytes and living skeletal muscle myotubes, contractile myofibrils were present in the same cell with non-contractile nascent myofibrils. The periodicities of small Z-bodies in the nascent non-contractile myofibrils were shorter than the Z-band spacings in the contractile myofibrils, yet both types of myofibrils contained muscle myosin. Over a period of 24 h, a nascent myofibril in a living, microinjected myotube was observed to grow from Z-body spacings of 0.9-1.3 micron to full sarcomere spacings (2.3 microns). During the same time, nascent myofibrils appeared de novo and Z-band alignment became more ordered in the fully formed myofibrils. Stress fibres were not observed to undergo the predictable type of growth seen in myofibrils, but stress fibre periodicities did change in some fibres; some shortened while others lengthened. The orientation of fibres shifted in cytoplasm of both mobile cells and stationary cells. Attachment plaques and foci also changed position and in some cases subdivided and/or disappeared. Models of stress fibres and polygonal networks are presented that suggest that the changes in the periodicities of the dense bodies in stress fibres and the distances between polygonal foci are related to the movement of the interdigitating actin and myosin filaments.

Actin Cytoskeleton↗
PubMed2005

Regulation of glioma cell migration by serine-phosphorylated P311.

P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59) near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation and induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311 and reduced glioma cell migration. Coimmunoprecipitation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, and immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of beta1 integrin function using TACbeta1A, a dominant-negative inhibitor of beta1 integrin signaling, suggesting that P311 acts downstream of beta1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Rac1 GTPase; small interfering RNA-mediated depletion of Rac1 suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility and invasion through the reorganization of actin cytoskeleton at the cell periphery.

Amino Acid Sequence↗