Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE J. FOXLABORATORYPROJECT SCIENTISTSTimothy O'Toole, Ph.D.RESEARCH ASSOCIATEKatarzyna Bialkowska, Ph.D.POSTDOCTORAL FELLOWSSucheta Kulkarni, Ph.D.Shan Wu, M.D.TECHNOLOGISTSHuiqin Nie, B.S.Andriy PodnolikovPetro RyobokonJoan E.B. Fox, Ph.D.Afirst step following integrin-inducedadhesion is the transmission of signalsthat lead to the extension of filopodiaand lamellipodia and the resultant spreading ofcells. Spreading cells are able to assemble bundlesof actin and myosin filaments and exertcontractile forces that allow them to migrate. Inspreading cells, contact with the extracellularmatrix is through complexes of integrin andsignaling molecules that continuously form andbreak down as actin filament networks formingbeneath the membrane cause the membrane’slamellipodia to extend.In more fully spread cells, integrin complexes,known as focal adhesions, are larger andinteract with the bundles of actin and myosinfilaments known as stress fibers. Focal adhesionsalso form and break down as cells migrate. Ourwork has focused on identifying mechanismsinvolved in the formation and breakdown ofintegrin complexes. We have identified a previouslyunrcognized type of integrin complex that formsprior to the focal complexes and focal adhesions:integrin clusters. Moreover, we have identifiedthree potential mechanisms whereby formation ofthese integrin clusters is regulated, one involvingcalpain, another the cytoskeletal protein skelemin,and another involving the SH3 domain of spectrin.Calpain, a Ca 2 -dependent protease presentin most cells, has two forms: one requiresmicromolar concentrations of Ca 2+ to beactivated, the other millimolar concentrations.Until recently, it was not known whether calpainis involved in signal transduction mechanisms,because these concentrations of calcium are notthought to be normally attained within cells.However, we have shown that calpain isactivated as a consequence of signaling acrossintegrins. We have demonstrated that themicromolar form of calpain is activated; itcleaves proteins that are present within theintegrin clusters. Moreover, we have observedBialkowska, K., Kulkarni, S., Du, X., Goll, D.E., Saido, T.C., and J.E.B. Fox (2000) Evidence thatβ3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clustersthat are distinct from focal complexes and focal adhesions that form as Rac and RhoA becomeactive. J. Cell Biol. 151:685-696.Reddy, K.B., Bialkowska, K., and J.E.B. Fox (2001) Dynamic modulation of cytoskeletalproteins linking integrins to signaling complexes in spreading cells: role of skelemin in initialintegrin-induced spreading. J. Biol. Chem. 276(30):28300-28308.Fox, J.E. (2001) Cytoskeletal proteins and platelet signaling. Thromb. Haemost. 86:198-213.Kulkarni, S., Goll, D.E., and J.E.B. Fox (2002) Calpain cleaves RhoA generating a dominantnegativeform that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol.Chem. 277:24435-24441.Bialkowska, K., Zaffran, Y., Meyer, S.C., and J.E.B. Fox (2003) 14-3-3zeta mediates integrininducedactivation of cdc42 and Rac: platelet glycoprotein Ib-IX regulates integrin-inducedsignaling by sequestering 14-3-3zeta. J. Biol. Chem. 278:33342-33350.The Department of Molecular CardiologySpectrin SH3 Domain, Calpain RegulateFormation of Focal Complexes inIntegrin-Induced Cell Spreadingthat calpain is present in the integrin clusters butnot in the focal complexes or adhesions. Previously,Rac1, a member of the family of RasGTPases, has been shown to be involved in theformation of focal complexes. We have shownthat calpain is essential for integrin clusterformation and for integrin-induced cell spreadingand that it acts upstream of Rac1. Thus, wepropose that calpain is a signaling moleculeinvolved in integrin-induced spreading ofadherent cells and that it acts early, prior toactivation of Rac1.Our studies on proteins that may berequired in integrin-induced signaling have alsoinvolved identifying cytoskeletal proteins thatinteract with the cytoplasmic domain of ligandoccupiedintegrin. Previously, we used the twohybridsystem to show that skelemin interactswith the cytoplasmic domain of β1- and β3-containing integrins. We have shown that thisinteraction occurs within spreading cells and thatskelemin interacts with integrins in integrinclusters, but not in focal complexes or in focaladhesions. We have identified the domains ofskelemin and integrin involved in the interactionand used this information to ablate the interactionand to show that skelemin plays an essential rolein allowing the very first steps of integrin clusterformation and cell spreading.Finally, we have investigated the potentialrole of the SH3 domain in spectrin. Previously,we showed that spectrin was present in integrincytoskeletalcomplexes in platelets. Using thetwo-hybrid system, we identified a proteincontaining a proline-rich region that interactedwith spectrin. We have now shown that bothspectrin and this protein are present in integrinclusters, but not in focal complexes or focaladhesions of spreading cells. Overexpression ofthe SH3 domain of spectrin and the proline-richdomain of the protein that interacts with spectrindid not affect the formation of integrin clustersbut prevented the formation of focal complexesand the spreading of cells.Our studies have focused on the mechanismsinvolved in integrin-induced signaling inspreading cells. We have identified an early typeof integrin cluster and shown that proteinspresent in these clusters include the Ca 2+ -dependent protease calpain, the cytoskeletalproteins skelemin and spectrin, and a putativesignaling molecule that contains a proline-richmolecule that interacts with spectrin’s SH3domain. All of these proteins are present inintegrin clusters but absent from focal complexesand focal adhesions. Moreover, calpain, spectrin,and the spectrin-interacting protein all appear toplay an essential role in regulating focal complexformation and integrin-induced cell spreading.122