Tiam1 is recruited to ß1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility.

14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing ß1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/ß1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into ß1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes.

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

BACKGROUND: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks. CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.

Adhesion-induced unclasping of cytoplasmic tails of integrin alpha(IIb)beta3.

Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.

Nuclear localization of Myomesin-1: possible functions.

Myomesin-I (also known as Skelemin) is a approximately 185 kDa protein, which is highly expressed in striated muscle. It contains the prototypic class-I (type-III fibronectin) and class-II (C2-immunoglobulin) motifs. Previous studies have shown the presence of Myomesin-I at the M-line of the sarcomere, where it is thought to interact with thick filament constituents. As reported previously, Myomesin-I was localized to the M-line in the adult cardiac myocytes (adult-myocytes). However, we found that Myomesin-I was also present exclusively in the nucleus of myocytes isolated from new born pups (neonatal-myocytes). In addition, the ectopically expressed Myomesin-I was primarily targeted to the nucleus, similar to the neonatal myocytes. Further investigations revealed that the nuclear-targeting signals were present within the N-terminal 256 residues. A strong consensus sequence for sumoylation is present within the N-terminal 256 residues and is implicated in the shuttling of Myomesin-I between nucleus and cytoplasm. Gene array analysis showed that the presence of Myomesin-I in the nucleus led to the differential expression of more than 42 genes. These studies show a novel and previously unknown localization and function for Myomesin-I.

Structural insight into the interaction between platelet integrin alphaIIbbeta3 and cytoskeletal protein skelemin.

Skelemin is a large cytoskeletal protein critical for cell morphology. Previous studies have suggested that its two-tandem immunoglobulin C2-like repeats (SkIgC4 and SkIgC5) are involved in binding to integrin beta3 cytoplasmic tail (CT), providing a mechanism for skelemin to regulate integrin-mediated signaling and cell spreading. Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin alphaIIbbeta3. Here, we show that skelemin IgC45 domains form a complex not only with integrin beta3 CT but also, surprisingly, with the integrin alphaIIb CT. Chemical shift mapping experiments demonstrate that both membrane-proximal regions of alphaIIb and beta3 CTs are involved in binding to skelemin. NMR structural determinations, combined with homology modeling, revealed that SkIgC4 and SkIgC5 both exhibited a conserved Ig-fold and both repeats were required for effective binding to and attenuation of alphaIIbbeta3 cytoplasmic complex. These data provide the first molecular insight into how skelemin may interact with integrins and regulate integrin-mediated signaling and cell spreading.

Integrative medicine: implementation and evaluation of a professional development program using experiential learning and conceptual change teaching approaches.

OBJECTIVE: To meet the increasing patient interest in complementary and alternative medicine (CAM), conventional physicians need to understand CAM, be willing to talk with their patients about CAM, and be open to recommending selected patients to appropriate CAM modalities. We aimed to raise physicians' awareness of, and initiate attitudinal changes towards CAM in the context of integrative medical practice. We developed and implemented a professional development program involving experiential learning and conceptual change teaching approaches. METHODS: A randomized controlled study with a pre-post design in a large academic medical center. The 8-hour intervention used experiential and conceptual change educational approaches. Forty-eight cardiologists were randomized to participant and control groups. A questionnaire measured physicians' conceptions of, and attitudes to CAM, the likelihood of changing practice patterns, and the factors most important in influencing such changes. The questionnaire included an embedded control question on a topic that was not the focus of this program. We administered the questionnaire before (pretest) and after (posttest) the intervention. We compared differences in pre- and post-intervention scores between the participant (N = 20) and control (N = 16) groups. We used both groups to identify factors that influenced their practice patterns. The study was NIH-funded and IRB-exempt. RESULTS: Both groups initially had little knowledge about, and negative attitudes to CAM. The participant group had significant positive changes in their conceptions about, and attitudes to CAM after the program, and significant improvements when compared with the control group. Participant physicians significantly increased in their willingness to integrate CAM in their practices. Physicians (combined groups) rated research evidence as the most important factor influencing their willingness to integrate CAM. They requested more research evidence for CAM efficacy, and more information on non-conventional pharmacology. Participants reflected enthusiasm for the experiential program. CONCLUSIONS: The participants were able to experience the positive effects of selected CAM modalities. It is possible to increase physician knowledge and change attitudes towards integrative medicine with an eight-hour intervention using experiential and conceptual change teaching approaches. PRACTICE IMPLICATIONS: Professional development on integrative medicine can be offered to medical practitioners using experiential learning and conceptual change teaching approaches, with the help of local CAM practitioners.

SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes.

In this study, we used cultured cells spreading on beta3 integrin substrates to examine the possibility that spectrin is involved in signal transduction. Spectrin clustered with specialized calpain-induced beta3 integrin signaling complexes that mediate the initial attachment of cells and initiate Rac activation and lamellipodia extension. It was absent from focal complexes and focal adhesions, the integrin complexes that mediate adhesion in lamellipodia and fully spread cells. Spectrin contains a Src homology (SH3) domain of unknown function. Cells overexpressing this domain adhered and calpain-induced integrin signaling complexes formed. However, Rac activation, lamellipodia extension and cell spreading were inhibited. Spreading was restored by overexpression of constitutively active Rac. These studies point to a previously unrecognized role for spectrin and its SH3 domain in initiating Rac activation in the specialized integrin clusters that initiate cell adhesion and spreading. Thus, spectrin may have a pivotal role in initiating integrin-induced physiological and pathological events such as development, proliferation, cell survival, wound healing, metastasis and atherosclerosis.

Megakaryocyte proliferation and ploidy regulated by the cytoplasmic tail of glycoprotein Ibalpha.

We have investigated the ability of glycoprotein (GP) Ibalpha, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3xi and to influence megakaryocytopoiesis. Using a Gp1ba(-/-) mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibalpha allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3xi. The observed phenotypes illustrate an involvement for GP Ibalpha in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibalpha/14-3-3xi/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibalpha cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.

14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta.

Integrin-induced cytoskeletal reorganizations are initiated by Cdc42 and Rac1 but little is known about mechanisms by which integrins activate these Rho GTPases. 14-3-3 proteins are adaptors implicated in binding and regulating the function and subcellular location of numerous signaling molecules. In platelets, the 14-3-3 zeta isoform interacts with the glycoprotein (GP) Ibalpha subunit of the adhesion receptor GP Ib-IX. In this study, we show that integrin-induced activation of Cdc42, activation of Rac, cytoskeletal reorganizations, and cell spreading were inhibited in Chinese hamster ovary cells expressing full-length GP Ibalpha compared with GP Ibalpha lacking the 14-3-3 zeta binding site. Activation of Rho GTPases and cytoskeletal reorganizations were restored by expression of 14-3-3 zeta. Spreading in cells expressing truncated GP Ibalpha was inhibited by co-expressing a chimeric receptor containing interleukin 2 receptor alpha and GP Ibalpha cytoplasmic domain. These results identify a previously unrecognized function of 14-3-3 zeta, that of mediating integrin-induced signaling. They show that 14-3-3 zeta mediates Cdc42 and Rac activation. They also reveal a novel function of platelet GP Ib-IX, that of regulating integrin-induced cytoskeletal reorganizations by sequestering 14-3-3 zeta. Signaling across integrins initiates changes in cell behavior such as spreading, migration, differentiation, apoptosis, or cell division. Thus, introduction of the 14-3-3 zeta binding domain of GP Ibalpha into target cells might provide a method for regulating integrin-induced pathways in a variety of pathological conditions.

Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion.

Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.

Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading.

Integrin-induced cell adhesion results in transmission of signals that induce cytoskeletal reorganizations and resulting changes in cell behavior. The cytoskeletal reorganizations are regulated by transient activation and inactivation of Rho GTPases. Previously, we identified mu-calpain as an enzyme that is activated by signaling across beta1 and beta3 integrins. We showed that it mediates cytoskeletal reorganizations in bovine aortic endothelial (BAE) and Chinese hamster ovary (CHO) cells and does so by acting upstream of Rac1 activation. Here we show that mu-calpain is also involved in inactivating RhoA during integrin-induced signaling. Cleavage of RhoA was detectable in BAE cells plated on an integrin substrate; it did not occur in cells plated on poly-l-lysine. Cleavage was inhibited by calpain inhibitors. In vitro, mu-calpain cleaved RhoA generating a fragment of the same size as in intact cells. The cleavage site was identified, an HA-tagged construct expressing calpain-cleaved RhoA generated, and the construct expressed in BAE and CHO cells. Calpain-cleaved RhoA inhibited integrin-induced stress fiber assembly and decreased cell spreading. Together, our data show that calpain cleaves RhoA and generates a form that inhibits integrin-induced stress fiber assembly and cell spreading.