Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

The Src family kinase Fyn mediates hyperosmolarity-induced Mrp2 and Bsep retrieval from canalicular membrane

In perfused rat liver, hyperosmolarity induces Mrp2- (Kubitz, R., D''urso, D., Keppler, D., and Häussinger, D. (1997) Gastroenterology 113, 1438–1442) and Bsep retrieval (Schmitt, M., Kubitz, R., Lizun, S., Wettstein, M., and Häussinger, D. (2001) Hepatology 33, 509–518) from the canalicular membrane leading to cholestasis. The aim of this study was to elucidate the underlying signaling events. Hyperosmolarity-induced retrieval of Mrp2 and Bsep from the canalicular membrane in perfused rat liver was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Both hyperosmotic transporter retrieval and Src kinase activation were sensitive to apocynin (300 μmol/liter), N-acetylcysteine (NAC; 10 mmol/liter), and SU6656 (1 μmol/liter). Also PP-2 (250 nmol/liter), which inhibited hyperosmotic Fyn but not Yes activation, prevented hyperosmotic transporter retrieval from the canalicular membrane, suggesting that Fyn but not Yes mediates hyperosmotic Bsep and Mrp2 retrieval. Neither hyperosmotic Fyn activation nor Bsep/Mrp2 retrieval was observed in livers from p47^phox knock-out mice. Hyperosmotic activation of JNKs was sensitive to apocynin and NAC but insensitive to SU6656 and PP-2, indicating that JNKs are not involved in transporter retrieval, as also evidenced by experiments using the JNK inhibitors L-JNKI-1 and SP6001255, respectively. Hyperosmotic transporter retrieval was accompanied by a NAC and Fyn knockdown-sensitive inhibition of biliary excretion of the glutathione conjugate of 1-chloro-2,4-dinitrobenzene in perfused rat liver and of cholyl-l-lysyl-fluorescein secretion into the pseudocanaliculi formed by hepatocyte couplets. Hyperosmolarity triggered an association between Fyn and cortactin and increased the amount of phosphorylated cortactin underneath the canalicular membrane. It is concluded that the hyperosmotic cholestasis is triggered by a NADPH oxidase-driven reactive oxygen species formation that mediates Fyn-dependent retrieval of the Mrp2 and Bsep from the canalicular membrane, which may involve an increased cortactin phosphorylation.     

Phosphorylation of Ser 21 in Fyn regulates its kinase activity,focal adhesion targeting,and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of highly cytokinergic IgE-induced mast cell adhesion by Src, Syk, Tec, and protein kinase C family kinases

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to Fc epsilon RI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive Fc epsilon RI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through beta1 and beta7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C theta as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C epsilon plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to Fc epsilon RI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion.     

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.     

Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.     

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

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

Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation.

pp125FAK is a tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125FAK is expressed as a separate protein, pp41/43FRNK. We have previously shown that overexpression of pp41/43FRNK inhibits tyrosine phosphorylation of pp125FAK and paxillin and, in addition, delays cell spreading and focal adhesion assembly. Thus, pp41/43FRNK functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125FAK promotes cell spreading. We report here that the inhibitory effects of pp41/43FRNK expression can be rescued by the co-overexpression of wild-type pp125FAK and partially rescued by catalytically inactive variants of pp125FAK. However, coexpression of an autophosphorylation site mutant of pp125FAK, which fails to bind the SH2 domain of pp60c-Src, or a mutant that fails to bind paxillin did not promote cell spreading. In contrast, expression of pp41/43FRNK and pp60c-Src reconstituted cell spreading and tyrosine phosphorylation of paxillin but did so without inducing tyrosine phosphorylation of pp125FAK. These data provide additional support for a model whereby pp125FAK acts as a "switchable adaptor" that recruits pp60c-Src to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly.     

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.     

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.     

The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading.

Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGF(TM)). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGF(TM) results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGF(TM) in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGF(TM)HA/Myc). HB-EGF(TM)HA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGF(TM)HA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGF(TM) shedding were identified as follows. (i) HB-EGF(TM)HA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGF(TM)HA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGF(TM) cleavage. (ii) Activation of an inducible Raf-1 kinase, DeltaRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGF(TM)HA/Myc within 20-40 min. (iii) Serum induced MAP kinase activation and HB-EGF(TM)HA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGF(TM)HA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.     

Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin alpha 4 beta 1.

We have recently reported the attachment and spreading of human umbilical vein endothelial cells (HUVECs) upon substrates containing covalently grafted Arg-Glu-Asp-Val (REDV) peptide (Hubbell, J. A., Massia, S. P., Desai, N. P., and Drumheller, P. D. (1991) Bio/Technology 9, 568-572). This peptide has been reported to be the minimal active sequence within the CS5 site of the alternatively spliced type III connecting segment (IIICS) region of fibronectin, and the integrin alpha 4 beta 1 has been identified as the receptor on melanoma cells for this site. The integrin alpha 4 beta 1 has also been identified as the receptor for the CS1 site in the IIICS region on cells of neural crest origin, melanoma cells, lymphocytes, and hematopoietic stem cells. In this study, we demonstrate that this integrin also serves as a receptor on HUVECs for the peptide REDV from the CS5 site. The alpha 4 subunit was shown to be expressed upon HUVEC membranes by whole-cell enzyme-linked immunosorbent assay. Antifunctional antibodies directed against integrin subunits alpha 4 and beta 1 inhibited cell adhesion on REDV-grafted substrates, but not on RGD-grafted substrates. The alpha 4 subunit localized into fibrillar structures within spread cells on the REDV-grafted substrates, but not within spread cells on RGD-grafted substrates. Two proteins (144 and 120 kDa) were isolated from HUVEC extracts by REDV ligand affinity chromatography and were demonstrated by immunoprecipitation and Western blot to be the integrin subunits alpha 4 (144 kDa) and beta 1 (120 kDa); furthermore, the immunoprecipitation analyses demonstrated that the subunits formed a complex. HUVEC binding to REDV-grafted substrates was inhibited by both soluble REDV and RGD, demonstrating that adhesion was biospecific and that the REDV peptide is RGD-like. In this report we demonstrate for the first time that alpha 4 is present in the endothelial cell membrane, in contrast to previous reports by others, and that integrin alpha 4 beta 1 is the receptor for REDV-mediated adhesion to the IIICS region of region of plasma fibronectin.     

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.     

The N termini of focal adhesion kinase family members regulate substrate phosphorylation,localization, and cell morphology

The focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta, PYK2, CADTK, RAFTK) are highly homologous FAK family members, yet clearly have unique roles in the cell. Comparative analyses of FAK and CAKbeta have revealed intriguing differences in their activities. These differences were investigated further through the characterization of a set of FAK/CAKbeta chimeric kinases. CAKbeta exhibited greater catalytic activity than FAK in vitro, providing a molecular basis for differential substrate phosphorylation by FAK and CAKbeta in vivo. Furthermore, the N terminus may regulate catalytic activity since chimeras containing the FAK N terminus and CAKbeta catalytic domain exhibited a striking high level of catalytic activity and substrate phosphorylation. Unexpectedly, a modulatory role for the N termini in subcellular localization was also revealed. Chimeras containing the FAK N terminus and CAKbeta C terminus localized to focal adhesions, whereas chimeras containing the N and C termini of CAKbeta did not. Finally, prominent changes in cell morphology were induced upon expression of chimeras containing the CAKbeta N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK family kinases.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

The toposome, essential for sea urchin cell adhesion and development, is a modified iron-less calcium-binding transferrin

We describe the structure and function of the toposome, a modified calcium-binding, iron-less transferrin, the first member of a new class of cell adhesion proteins. In addition to the amino acid sequence of the precursor, we determined by Edman degradation the N-terminal amino acid sequences of the mature hexameric glycoprotein present in the egg as well as that of its derived proteolytically modified fragments necessary for development beyond the blastula stage. The approximate C-termini of the fragments were determined by a combination of mass spectrometry and migration in reducing gels before and after deglycosylation. This new member of the transferrin family shows special features which explain its evolutionary adaptation to development and adhesive function in sea urchin embryos: (i) a protease-inhibiting WAP domain, (ii) a 280 amino acid cysteine-less insertion in the C-terminal lobe, and (iii) a 240 residue C-terminal extension with a modified cystine knot motif found in multisubunit external cell surface glycoproteins. Proteolytic removal of the N-terminal WAP domain generates the mature toposome present in the oocyte. The modified cystine knot motif stabilizes cell-bound trimers upon Ca-dependent dissociation of hexamer-linked cells. We determined the positions of the developmentally regulated cuts in the cysteine-less insertion, which produce the fragments observed previously. These fragments remain bound to the hexameric 22S particle in vivo and are released only after treatment of the purified toposome with reducing agents. In addition, some soluble smaller fragments with possible signal function are produced. Sequence comparison of five sea urchin species reveals the location of the cell-cell contact site targeted by the species-specific embryo dissociating antibodies. The evolutionary tree of 2-, 1-, and 0-ferric transferrins implies their evolution from a basic cation-activated allosteric design modified to serve multiple functions.     

Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity

The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.     

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell–matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82’s contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.