Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIgamma661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIgamma661 and Src. The phosphorylation of Y644 results in an approximately 15-fold increase in binding affinity to the talin head domain and blocks beta-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a "molecular switch" for talin binding between PIPKIgamma661 and beta-integrin that may regulate dynamic FA turnover.     

Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.     

Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.     

Type I gamma phosphatidylinositol phosphate kinase is required for EGF-stimulated directional cell migration

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) modulates a plethora of cytoskeletal interactions that control the dynamics of actin assembly and, ultimately, cell migration. We show that the type Igamma phosphatidylinositol phosphate kinase 661 (PIPKIgamma661), an enzyme that generates PI4,5P(2), is required for growth factor but not G protein-coupled receptor-stimulated directional migration. By generating PI4,5P(2) and regulating talin assembly, PIPKIgamma661 modulates nascent adhesion formation at the leading edge to facilitate cell migration. The epidermal growth factor (EGF) receptor directly phosphorylates PIPKIgamma661 at tyrosine 634, and this event is required for EGF-induced migration. This phosphorylation regulates the interaction between PIPKIgamma661 and phospholipase Cgamma1 (PLCgamma1, an enzyme previously shown to be involved in the regulation of EGF-stimulated migration). Our results suggest that phosphorylation events regulating specific PIPKIgamma661 interactions are required for growth factor-induced migration. These interactions in turn define the spatial and temporal generation of PI4,5P(2) and derived messengers required for directional migration.     

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex,as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.     

Rac1 and calmodulin interactions modulate dynamics of ARF6-dependent endocytosis

The main cellular Ca(2+) sensor, calmodulin (CaM), interacts with and regulates several small GTPases, including Rac1. The present study revealed high binding affinity of Rac1 for CaM and uncovered two new essential binding domains in Rac1: the polybasic region, important for phosphatidylinositol-4-phosphate 5-kinase (PIP5K) interaction, and the adjacent prenyl group. CaM inhibition increased Rac1 binding to PIP5K and induced an extensive phosphatidylinositol 4,5-bisphosphate (PI4,5P(2) )-positive tubular membrane network. Immunofluorescence demonstrated that the tubules were plasma membrane invaginations resulting from an ADP-ribosylation factor 6 (ARF6)-dependent and clathrin-independent pathway. The role of Rac1 in this endocytic route was analyzed by expressing constitutively active and inactive mutants. While active Rac1 impaired tubulation, the inactive mutant enhanced it. Intriguingly, inactive mutant expression elicited tubulation by recruiting PIP5K and inhibiting Rac1 at the plasma membrane. Accordingly, CaM inhibition inactivated Rac1 and increased Rac1/PIP5K interaction. Therefore, our findings highlight an important new role for Rac1 and CaM in controlling clathrin-independent endocytosis.     

GRP1 pleckstrin homology domain: activation parameters and novel search mechanism for rare target lipid

Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P(3) (or PIP(3)) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP(3) signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP(3). A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K(D) of 50 +/- 10 nM for GRP1-PH domain binding to membrane-bound PIP(3) in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K(D) lies in a suitable range for regulation by physiological PIP(3) signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP(3). Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP(3) headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.     

The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.     

The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.     

Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.     

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP₂ is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP₂ in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP₂ increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP₂. In contrast, PIP5Kα knockdown that reduced PIP₂ levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP₂-dependent recruitment of TIRAP to the plasma membrane.     

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.     

Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.     

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.Receptor tyrosine kinase (RTK) signaling is initiated on binding of soluble growth factors to growth factor receptors such as the insulin receptor (IR) or epidermal growth factor receptor (EGFR), or on binding of membrane-bound ephrins, as is the case for Eph receptors. Intracellular signaling is then propagated through PTMs, which commonly serve to regulate protein function by acting as docking sites for recruitment of modular protein interaction domains. Phosphorylation is the best studied PTM, and is a principle mechanism regulating intracellular signaling.A common element in RTK signaling involves autophosphorylation of the intracellular portion of the receptor (Fig. 1). RTKs become activated as a result of ligand-stabilized dimerization or oligomerization. For instance, in the EGFR subfamily (which includes ErbB and EGF receptors), the formation of homo- or heterodimers is initiated by ligand binding and subsequent exposure of a dimerization domain (Hynes and Lane 2005). Dimerization of the RTKs allows autophosphorylation of the RTKs; EGFR is exceptional in that an allosteric interaction between the kinase domains of adjacent monomers is responsible for the receptor activation (Zhang et al. 2006). However, in the majority of cases dimerization enhances RTK catalytic activity through phosphorylation of the kinase activation loop, and in some instances the juxtamembrane region, and recruits signaling effectors through the creation of pTyr docking sites. The specific interaction of signaling proteins with these pTyr-binding motifs activates signaling pathways, such as canonical signaling through the Ras-mitogen activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)-Akt, and phospholipase C-gamma (PLC-γ) pathways. These RTK pathways can result in a variety of cellular processes, including differentiation, proliferation, survival, and migration (Fig. 1). The cellular context of signaling can dictate the biological outcome, and how each RTK initiates a given cellular process remains an area of active research.Open in a separate windowFigure 1.Receptor tyrosine kinases activate downstream pathways through recruitment of proteins containing pTyr-binding domains. Receptor tyrosine kinases are activated on growth factor binding to the extracellular domain of the receptor, leading to receptor dimerization and tyrosine phosphorylation (yellow circles labeled with a P) of their cytoplasmic tails, which act as docking sites for recruitment of PTB and SH2 domains. Various RTKs can mediate a diverse set of cellular processes (colored boxes) determined by the recruitment of specific SH2- and PTB-domain-containing proteins. The gray box displays how the adaptor Grb2 is recruited to an RTK through recognition of the pY-x-N (pY = pTyr, x = any natural amino acid) and activates cell growth and survival pathways such as MAPK and AKT, respectively, through complex formation via its SH3 domains.Tyrosine phosphorylation mediates RTK signaling through the recruitment and activation of proteins involved in downstream signaling pathways, mediated through pTyr binding of the SH2 and PTB domains of signaling effectors. SH2 and PTB domains are found in an otherwise diverse set of proteins containing a range of distinct catalytic and interaction domains, and provide a degree of specificity through their recognition of both a pTyr residue and surrounding amino acids. Here we will discuss the properties of proteins that contain SH2 and PTB domains and their roles in signaling downstream of RTKs, as well as the mechanisms by which they regulate the activity of these signaling effectors.     

Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP(2)), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP(2) for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP(2) derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP(2) substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP(2) analogues effectively function as substrates of PLC-δ1, and the K(M) and V(max) values obtained with one of these are similar to those observed with native PIP(2) substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors.     

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.     

Vinculin activates inside-out signaling of integrin αIIbβ3 in Chinese hamster ovary cells

Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.