Patent Application: US-13779508-A

Abstract:
the invention provides a method of inducing neurofibromatosis type 2 in schwann cells . the method comprises contacting the cells with laminin - 1 so as to bind α6β1 integrin sufficiently to activate endogenous kinase cdc42 - pak ; and phosphorylating schwannomin - s518 in the cells by the activated kinase , effectively inactivating schwannomin &# 39 ; s tumor suppressor activity and allowing proliferation of subconfluent schwann cells , thereby modeling nf2 . the invention also includes a method of preventing a schwann cell from forming a tumor by contacting the cell with an amount of tyrphostin ag825 sufficient to inhibit a receptor selected from erb2 , erb3 , β1 integrins and combinations thereof , so as to prevent phosphorylation of schwannomin - s815 by one or more endogenous kinases .

Description:
the present invention will now be described more fully hereinafter with reference to the accompanying drawings , in which preferred embodiments of the invention are shown . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . any publications , patent applications , patents , or other references mentioned herein are incorporated by reference in their entirety . in case of conflict , the present specification , including any definitions , will control . in addition , the materials , methods and examples given are illustrative in nature only and not intended to be limiting . accordingly , this invention may be embodied in different forms and should not be construed as limited to the illustrated embodiments set forth herein . rather , these illustrated embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . abbreviations used herein are as follows : cyclic - amp response element binding protein ( p - creb ); neurofibromatosis type 2 ( nf2 ); p21 - activated kinase ( pak ); neuregulin - 1 ( nrg1 ); protein kinase a ( pka ); schwann cells ( scs ); and schwannomin ( sch ). the neurofibromatosis type 2 ( nf2 ) tumor suppressor schwannomin ( sch ), also known as merlin , is a membrane - cytoskeleton linking protein ( rouleau et al ., 1993 ; trofatter et al ., 1993 ). mutations in the nf2 gene predispose individuals to benign , slow - growing schwannomas . sch &# 39 ; s conformation , localization , and phosphorylation are important determinants of its ability to regulate proliferation and actin organization ( reviewed in mcclatchey and giovannini , 2005 ). the tumor suppressor function of sch is associated with its closed , intracellular form lacking phosphorylation on serine 518 ( s518 ; rong et al ., 2004 ; shaw et al ., 2001 ). in this conformation , sch inhibits rac - mediated signaling cascades and progression through the g1 phase of the cell cycle ( reviewed in okada et al ., 2007 ). phosphorylation of sch - s518 is believed to stabilize sch in the open conformation , inhibiting its tumor suppressor function while unmasking binding sites for trans - membrane receptors and actin - associated proteins ( james et al ., 2001 ; rong et al ., 2004 ). p21 - activated kinase ( pak ) and protein kinase a ( pka ) phosphorylate sch on s518 , but the receptor mechanisms leading to sch phosphorylation are unknown ( kissil et al ., 2002 ; xiao et al ., 2002 ; alfthan et al ., 2004 ). previously , we demonstrated that sch localization to the plasma membrane , and its phosphorylation on s518 requires direct binding of residues 50 - 70 within sch &# 39 ; s n - terminus to the scaffold protein , paxillin ( fernandez - valle et al ., 2002 , thaxton et al ., 2007 ). we also demonstrated that sch is present at the plasma membrane of subconfluent schwann cells ( scs ), where sch and paxillin interact with β1 integrin and erbb2 ; receptors critical for sc adhesion , motility , proliferation and myelination ( fernandez - valle et al ., 2002 ; reviewed in garratt et al ., 2000 ; reviewed in chernousov and carey , 2000 ). paxillin also recruits pak to the plasma membrane , and serves as a scaffold for sch - pak interactions by binding multiple regulators of rac and cdc42 , members of the rho family of gtpases , as well as actin binding proteins and components of focal complexes and adhesions ( reviewed in turner , 2000 ). here , we demonstrate that nrg1 and laminin - 1 , ligands for erbb and β1 integrin receptors , respectively , induce sch phosphorylation in scs through two independent pathways , nrg1 by pka , and laminin by pak . nrg1 and laminin - 1 do not synergize to increase sch phosphorylation , but rather nrg1 through pka partially antagonizes laminin - induced , and pak - mediated sch phosphorylation . these findings show that sch is a convergence point for transduction of signals from erbb and β1 integrin receptors that regulate proliferation , differentiation and cytoskeletal dynamics in scs during peripheral nerve development . the following materials were used : natural mouse laminin - 1 and lipofectamine 2000 ( invitrogen ; carlsbad , calif ., usa ), ag825 and pki 14 - 22 amide ( emd biosciences ; san diego , calif ., usa ). sch - gfp constructs were described previously ( thaxton et al ., 2007 ). recombinant human nrg - 1 beta / type ii was a generous gift from mark marchionni . myc - pak k299a and myc - pak l107f . t423e ( myc - pak t423e ) constructs were generous gifts from gary bokoch ( the scripps research inst ., la jolla , calif ., usa ). antibodies were purchased from the following sources : erbb2 and sch ( c18 ) from santa cruz biotechnology ( santa cruz , calif ., usa ); β1 integrin from bd transduction labs ( san jose , calif ., usa ); pak , myc , and p - creb from cell signaling ( boston , mass ., usa ); ps518 - sch and p - erbb2 ( y1248 ) from abcam ( cambridge . mass ., usa ); p - pak ( t423 ) from rockland immunologicals , inc . ( gilbertsville , pa ., usa ); xpress from invitrogen ; erbb2 from emd biosciences ; and alexa fluor conjugated secondary antibodies from invitrogen . primary rat sc cultures were prepared from neo - natal day 1 rat pups as described previously ( chen et al ., 2000 ). subconfluent sc cultures were grown on glass coverslips coated with poly - l - lysine ( pll ; 200 mg / ml ) alone or sequentially with laminin - 1 ( 25 mg / ml ). cultures were starved overnight in dulbecco &# 39 ; s modified eagle medium ( dme ) with 0 . 5 % fetal bovine serum ( d0 . 5 ) before use . the scs were either left unstimulated , or were stimulated with nrg ( 10 ng / ml ) for 30 minutes . for laminin - 1 stimulation , primary rat scs were grown on pll coated glass coverslips and were starved overnight in d0 . 5 . the scs were then stimulated with soluble laminin - 1 ( 10 mg / ml ) or were left unstimulated . the scs were immunostained as described previously ( fernandez - valle , 2002 ). cells were analyzed with a zeiss laser scanning microscope and lsm 510 software . images shown in each figure are single planes , and were collected with identical settings , and were processed identically . primary rat scs were grown to approximately 60 % confluency on pll coated dishes . the cultures were serum starved overnight in d0 . 5 and were either left in d0 . 5 or were pre - incubated with ag825 ( 1 mm ) or pki 14 - 22 ( 50 nm ) for 1 hour . next , the scs were either left unstimulated or were stimulated with nrg1 ( 10 ng / ml ) and / or laminin - 1 ( 10 mg / ml ) in the presence and absence of ag825 ( 1 mm ) or pki 14 - 22 ( 50 nm ) for 30 minutes . the scs were extracted as described previously ( fernandez - valle et al ., 2002 ) in either tan buffer ( 10 mm tris - acetate ph8 . 0 , 100 mm nacl , and 1 % igepal ) or hepes buffer ( 50 mm hepes , 1 mm dtt , 150 mm nacl , 1 % igepal ) containing protease inhibitors . following extraction , the sc lysate was measured for protein concentration , and 10 mg of total sc lysate was separated by sds - page and was transferred to pvdf membranes . the indicated primary antibodies were used , followed by corresponding hrp - conjugated secondary antibody and chemiluminescence detection . densitometric analysis was conducted on all western blots . bands intensities were quantified and normalized to gapdh , and to their respective total proteins for phosphorylated forms . statistical analysis was acquired using the student t - test by paired analysis . subconfluent sc cultures grown in medium containing 10 % fbs , forskolin ( 2 mm ) and pituitary extract ( 20 mg / ml ) were extracted in tan buffer and 500 mg of lysate were immunoprecipitated with β1 integrin antibody , as described previously ( chen et al ., 2000 ). immunoprecipitation with 31 integrin antibody covalently linked to magnetic beads was performed as described previously ( taylor et al ., 2003 ). primary rat sc cultures were transfected using lipofectamine 2000 as described previously ( thaxton et al ., 2007 ). thirty - six hours after transfection , the scs were immunostained . nrg and laminin induce phosphorylation of sch at sc distal tips and radial membrane protrusions nrg and laminin activate cdc42 / rac gtpases and pak in other cell types ( adam et al ., 1998 ; del pozo et al ., 2000 ). work from this laboratory has demonstrated that sch can interact with both erbb2 and β1 integrin , and that paxillin - dependent localization to the plasma membrane is required for phosphorylation of sch by cdc42 - pak ( fernandez - valle et al ., 2002 ; thaxton et al . 2007 ). here , we sought to identify receptor ( s ) that trigger pak activity and sch phosphorylation . we stimulated subconfluent and serum - starved primary rat scs grown on laminin - 1 with nrg1 for 30 minutes , and assessed the phosphorylation states and localization of erbb2 , sch and pak . erbb2 , erbb3 , and sch were found along sc processes , and were concentrated at the distal tips . nrg1 stimulation induced a focal enrichment of p - erbb2 and ps518 - sch at the distal tips of sc processes and within membrane protrusions ( fig1 ). these molecules co - localized with cdc42 and paxillin . phosphorylated pak was also enriched in membrane protrusions and at the distal tips where it co - localized with erbb2 . we quantified the changes in phosphorylation of erbb2 , sch , and pak in serum starved and nrg1 stimulated sc processes ( fig2 a , b ). we found marked increases in fluorescence intensity along sc processes for p - erbb2 , ps518 - sch , and p - pak , particularly at process tips of stimulated versus starved scs ( fig2 a ). p - erbb2 levels increased by 1 . 6 - fold , ps518 - sch by 1 . 5 - fold , and p - pak by 1 . 9 - fold in nrg1 stimulated versus starved scs ( fig2 b ). these results demonstrate that acute stimulation of scs with nrg1 induces phosphorylation of sch downstream of erbb2 / erbb3 , possibly by pak . as the scs were grown on laminin - 1 , a ligand for β1 integrin that mediates sc adhesion ( fernandez - valle et al ., 1994 ), we tested whether a 30 - minute exposure to soluble laminin - 1 stimulated sch phosphorylation in subconfluent and serum - starved scs grown on pll . laminin - 1 promoted a strong increase in sch and pak phosphorylation compared to starved scs ( fig2 c ). quantification of fluorescence intensity along the processes revealed a 1 . 4 - fold increase in ps518 - sch and a 1 . 7 - fold increase in p - pak compared to untreated scs ( fig2 d ). these results demonstrate that adhesion to laminin - 1 induces sch phosphorylation at the plasma membrane , possibly by pak . to determine the relative contributions of erbb2 / erbb3 and β1 integrin activation on sch phosphorylation , we repeated the experiments using scs plated on pll rather than laminin - 1 , and employed the use of ag825 to specifically inhibit erbb2 kinase activity ( osherov et al ., 1993 ). subconfluent scs were starved and then were stimulated with nrg1 in the presence and absence of ag825 ( fig3 ). nrg1 promoted a 4 . 8 - fold increase in p - erbb2 and a 2 . 7 - fold increase in ps518 - sch compared to starved scs . ag825 reduced nrg1 stimulated phosphorylation of erbb2 significantly , as well as sch - s518 phosphorylation ( fig3 a , b ). surprisingly , no pak phosphorylation was observed in nrg1 stimulated scs . when total protein levels were assessed , we found that stimulation with nrg1 reduced the amount of erbb2 by 50 % ( fig3 c , d ). this is consistent with rapid , ligand - induced degradation of erbb2 receptors ( lotti et al ., 1992 ). ag825 attenuated the reduction in erbb2 levels . these results suggest that nrg1 triggers sch phosphorylation independently of pak in scs . pka has been reported to phosphorylate sch at s518 in vitro ( alfthan et al ., 2004 ). additionally , nrg1 stimulation has been suggested to induce pka activity in scs ( kim et al ., 1997 ). therefore , we tested whether pka phosphorylated sch in response to nrg1 activation of erbb2 / erbb3 receptors on scs . subconfluent scs were serum starved and were stimulated with nrg1 in the presence and absence of pki 14 - 22 amide , a specific inhibitor of pka activity ( fig3 e , f ). nrg1 alone stimulated a 2 . 1 - fold increase in phosphorylated sch compared to starved scs , and induced phosphorylation of the cyclic - amp response element binding protein ( p - creb ), a known substrate for pka . pki 14 - 22 reduced the levels of sch and creb phosphorylation to near basal levels observed in starved controls . additionally , stimulation of starved scs for 30 minutes with forskolin , an activator of adenylyl cyclase that increases intracellular cyclic - amp and activates pka , also induced phosphorylation of sch and creb . these results provide evidence for pka - dependent phosphorylation of sch following nrg1 binding to erbb2 / erbb3 in scs . to determine if laminin - 1 induced phosphorylation of sch by pak , we stimulated scs grown on pll with soluble laminin - 1 for 30 minutes and conducted western blot analyses ( fig4 ). laminin - 1 promoted a substantial 3 . 5 - fold increase in sch - s518 phosphorylation and a 1 . 5 - fold increase in pak phoshorylation compared to starved scs ( fig4 a , b ). the levels of total sch and β1 integrin did not significantly change in response to laminin - 1 , while the levels of total pak fell by 25 % ( fig4 c , d ). we additionally tested whether laminin - 1 activated erbb2 and pka . laminin - 1 stimulated scs had no change in the basal level of p - erbb2 and did not contain p - creb ( data not shown ). these findings are consistent with pak mediated phosphorylation of sch - s518 following laminin binding to β1 integrins expressed on the surface of subconfluent scs . to obtain additional evidence that pak phosphorylates sch in response to stimulation with laminin - 1 , we transiently transfected scs plated on pll and laminin - 1 with gfp - tagged sch and myc - tagged pak kinase mutant constructs ( fig5 ). as shown previously , expression of wild - type sch ( sch - gfp ) resulted in sch phosphorylation at the plasma membrane within discrete membrane protrusions that contain p - pak ( thaxton et al ., 2007 ). co - expression of sch - gfp with catalytically inactive pak ( myc - pak k299a ) resulted in a loss of ps518 - sch fluorescence in these domains and throughout the sc , whereas co - expression with constitutively active pak ( myc pak t423e ) resulted in unrestricted sch phosphorylation . these results support pak - mediated phosphorylation of sch induced by β1 integrin adhesion to laminin - 1 . β1 integrin and erbb2 exist as a functional co - receptor complex on the sc surface to investigate the possibility that erbb2 and β1 integrin act as co - receptors to regulate sch phosphorylation , we tested their ability to co - localize and co - immunoprecipitate . we found that β1 integrin and erbb2 co - localized with ps518 - sch and p - erbb2 at the distal tips of sc processes acutely stimulated with nrg1 ( fig6 a ). β1 integrin immunoprecipitations prepared from lysates of subconfluent sc cultures contained β1 integrin and erbb2 , as well as , ps518 - sch and paxillin ( fig6 b ). to ascertain whether the receptors associated on the cell surface , ( 31 integrins were clustered on suspended intact scs using a β1 integrin antibody immobilized on magnetic beads and were subsequently lysed and the clustered receptor complexes were isolated ( fig6 c ). a subset of erbb2 receptors and ps518 - sch were present in the β1 integrin immunoprecipitate . rhoa , used as a control , was not present in the immunoprecipitate . trans - activation of integrins and receptor tyrosine kinases occurs , and can produce changes in receptor protein expression ( reviewed in lee and juliano , 2004 ). to determine if this occurs in scs , β1 integrin and erbb2 protein levels were measured following stimulation with nrg1 or laminin - 1 for 30 minutes . stimulation with nrg1 significantly increased β1 integrin levels 2 . 3 - fold , but decreased erbb2 levels by half as compared to starved scs ( fig6 d , e ). stimulation of scs with nrg1 and ag825 suppressed nrg1 &# 39 ; s effect on both β1 integrin and erbb2 protein levels . stimulation of scs with laminin - 1 did not alter β1 integrin protein levels , but did increase erbb2 protein levels by a statistically significant 1 . 3 - fold compared to untreated scs ( fig6 f , g ). surprisingly , stimulation with laminin - 1 and ag825 decreased β1 integrin levels by 50 %, but did not alter erbb2 levels with respect to laminin - stimulated scs . this is consistent with a basal level of autocrine activation of erbb2 / erbb3 in scs . scs have been shown to synthesize and secrete nrgs in response to serum deprivation , thereby transducing survival signals ( rosenbaum et al ., 1997 ). dual stimulation with nrg and laminin does not synergistically increase sch phosphorylation to determine if simultaneous activation of erbb2 / erbb3 and β1 integrin in scs synergize to increase phosphorylation of sch , we stimulated serum - starved scs grown on pll for 30 minutes with nrg1 and soluble laminin - 1 , in the presence and absence of ag825 ( fig7 ). dual stimulation resulted in a significant 1 . 8 - fold increase in ps518 - sch and a 2 . 1 - fold increase in p - pak compared to unstimulated scs . this level of sch phosphorylation was not higher than levels observed in scs stimulated with either nrg1 or laminin - 1 alone , which were 2 . 7 - fold and 3 . 5 - fold higher than starved scs , respectively ( fig7 a , b ). surprisingly , stimulation with nrg1 and laminin - 1 in the presence of ag825 promoted greater phosphorylation of sch - s518 ( a 4 . 6 - fold increase ) and pak ( a 3 . 1 - fold increase ) compared to starved scs , suggesting that erbb2 kinase activity partially inhibits pak and its phosphorylation of sch . pka has been shown to directly phosphorylate and inhibit pak ( howe and juliano , 2000 ). we found that ag825 inhibited phosphorylation of creb in response to nrg1 in dually stimulated scs , consistent with pka inhibition of pak ( fig7 g ). sch , pak and β1 integrin protein levels were not significantly changed in scs stimulated with nrg1 and laminin - 1 in the presence and absence of ag825 ( fig7 c , d ). stimulation with nrg1 and laminin - 1 resulted in a 30 % decrease in erbb2 compared to starved scs . this result indicates that pka activated downstream of erbb2 kinase activity partially inhibits pak - dependent phosphorylation of sch in response to laminin - 1 . a model consistent with our results is shown ( fig8 ). phosphorylation of s518 is a critical switch that controls sch &# 39 ; s tumor suppressor activity . pak and pka have been shown to phosphorylate sch on s518 when overexpressed with sch in cell lines and in in vitro kinase assays , but neither kinase has been linked to receptor activation and phosphorylation of endogenously expressed sch in any cell type ( kissil et al ., 2002 ; xiao et al ., 2002 ; alfthan et al ., 2004 ). here , we identify two receptors that lead to rapid phosphorylation of sch - s518 in scs . laminin - 1 binding to β1 integrin activates pak , whereas nrg1 binding to erbb receptors activates pka . each kinase phosphorylates sch - s518 within 30 minutes of stimulation . both receptors regulate all stages of schwann cell development including proliferation , and both play central roles in the tumorigenic and metastatic capacities of many additional cell types . our data provide strong evidence that nrg1 binding to erbb2 / erbb3 induces pka - dependent phosphorylation of endogenous sch . this conclusion is supported by the following results . first , serum - starved scs have basal levels of phosphorylated pak and sch . nrg1 inhibits basal pak activity while increasing the amount of phosphorylated sch , 2 . 7 - fold . second , inhibition of erbb2 kinase activity by ag825 reduces sch - s518 phosphorylation in response to nrg1 by 50 %. third , the pka inhibitor , pki 14 - 22 similarly reduces sch - s518 phosphorylation in response to nrg1 by 70 %. lastly , although not as effective as nrg1 , forskolin increases sch phosphorylation . we also show that nrg1 promotes phosphorylation of creb and that both ag825 and pki 14 - 22 inhibit this phosphorylation , consistent with nrg stimulation of pka activity . in support , others have also found evidence of pka activation by nrg in scs ( kim et al ., 1997 ). overall , our results indicate that nrg binds to erbb2 / erbb3 receptors and stimulates rapid phosphorylation of sch on s518 by pka . cell adhesion to extracellular matrix through integrins activates pak ( del pozo et al ., 2000 ). laminin - 1 is present in the endoneurium of nerves in perinatal mice , and promotes strong in vitro adhesion , migration and proliferation of scs ( milner et al ., 1997 ; dubovy et al ., 2000 ). previously we demonstrated that α6β1 integrin is the predominant laminin - 1 binding integrin present in scs at this stage of development ( fernandez - valle et al ., 1994 ). our new findings indicate that laminin - 1 binding to α6β1 integrin promotes sch - s518 phosphorylation by pak . our evidence is as follows : first , stimulation of scs with soluble laminin - 1 increases both p - pak ( 1 . 5 - fold ) and ps518 - sch ( 3 . 5 - fold ) over basal levels . similarly , p - pak and ps518 - sch are increased within sc processes as assessed by quantification of immunofluorescence . second , sch - gfp expressed in scs adhering to laminin - 1 is phosphorylated by an endogenous kinase , predominantly when localized at the plasma membrane of cellular processes and particularly in radial membrane protrusions . previously , we reported that cdc42 - pak rather than rac - pak was associated with phosphorylation of sch in these domains ( thaxton et al ., 2007 ). consistently , we find that expression of catalytically inactive pak inhibits phosphorylation of sch - gfp in scs adhering to laminin - 1 . lastly , laminin - 1 does not activate pka or trans - activate erbb2 , as p - erbb2 and p - creb were not found , ruling out pka dependent phosphorylation of sch in response to laminin - 1 . it has been established that pak is recruited to focal complexes through an indirect interaction with paxillin , stimulated by cdc42 and rac activity ( brown et al ., 2002 ). together , our results indicate that aggregation of α6β1 integrins by laminin - 1 triggers translocation of a pak - paxillin - sch complex to nascent focal complexes where sch - s518 is phosphorylated by cdc42 - pak . our data demonstrate that erbb2 / erbb3 and β1 integrin physically interact and function as co - receptors that regulate both the turnover rate of each receptor and their downstream signals . erbb2 / erbb3 and β1 integrin co - immunoprecipitate and co - localize on the sc surface and are enriched at the distal tips of sc processes stimulated with nrg1 . simultaneous activation of erbb2 / erbb3 and β1 integrin receptors does not synergistically increase sch phosphorylation , but rather erbb2 activity appears to antagonize pak - dependent phosphorylation of sch . in scs stimulated with nrg1 , laminin - 1 and ag825 , ps518 - sch and p - pak levels increase over dually stimulated scs while p - creb is eliminated . ag825 , in the absence of nrg1 also increases basal levels of phosphorylated pak and sch ( data not shown ), consistent with autocrine stimulation of erbb2 and pka activity ( rosenbaum et al ., 1997 ). pka has been shown to directly phosphorylate and inhibit pak in nih3t3 cells ( howe and juliano , 2000 ). together , these findings indicate that erbb2 , through pka , antagonizes pak dependent phosphorylation of sch downstream of β1 integrin . β1 integrin and erbb receptors regulate sc proliferation during development . conditional inactivation of genes encoding their respective ligands , the laminin g1 gene and the neuregulin gene , in mice are associated with low proliferative capacity of scs during development , demonstrating that both receptors are essential for sc proliferation ( reviewed in garratt et al ., 2000 ; chen and strickland , 2003 ; yang et al ., 2005 ). cooperation between receptor tyrosine kinase and integrin signaling is required for activation of the ras - raf - mek - erk pathway , which is active in both human and rodent scs ( reviewed in lee et al ., 2004 ; monje et al ., 2006 ). activation of mitogenic receptor tyrosine kinases , in the absence of integrin - dependent adhesion , is coupled only to ras and raf and does not lead to mek and erk activity . adhesion of integrins to extracellular matrix activates the rho family of gtpases and pak , allowing pak phosphorylation of c - raf and mek . mek then associates with , and phosphorylates erk ( del pozo et al ., 2000 ; coles and shaw 2002 ). one mechanism by which sch restricts proliferation is by inhibiting rac - pak activity in confluent cells ( reviewed in okada et al ., 2007 ). phosphorylation of sch downstream of both erbb and β1 integrin receptors would inactivate this ability and would allow rac - pak signaling to couple to ras - erk pathways and stimulate proliferation of subconfluent cells . consistent with the rapid turnover of focal contacts in subconfluent , motile cells , we find that β1 integrin and erbb2 protein levels are rapidly modulated by receptor activity . nrg1 has a greater effect on erbb2 and β1 integrin levels than laminin - 1 . whereas laminin - 1 stimulates a 30 % increase in erbb2 protein expression over starved scs , nrg1 promotes a 50 % decrease in erbb2 receptors while increasing β1 integrin levels 2 . 3 fold over starved scs . ag825 attenuates the loss of erbb2 protein in response to nrg1 and inhibits the increase in β1 integrin , confirming that erbb2 kinase activity modulates the fate of each receptor . in laminin - 1 stimulated scs , ag825 decreases β1 integrin levels , consistent with autocrine stimulation of erbb2 in scs . these effects are likely mediated through changes in protein stability and / or degradation , as they occur within 30 minutes of stimulation . moreover , our results show that each receptor has a different fate after activation , β1 integrins are stabilized following nrg1 stimulation , whereas erbb2 is degraded . this also implies that erbb2 dependent inhibition of pak is transient , and that β1 integrin and pak dependent phosphorylation of sch occurs during the time erbb2 receptor expression on the plasma membrane is low . sch is at a critical convergence point for transduction of signals from these receptors , and reveals why loss of this protein in scs predisposes them to tumor formation . there is evidence that sch controls endocytosis of erbb family members , possibly through its interaction with hrs ( scoles et al ., 2005 ; maitra et al ., 2006 ). additionally , other paxillin binding proteins regulate vesicle trafficking and receptor degradation ( reviewed in turner , 2000 ). of note , human schwannoma cells have increased expression of β1 integrin and activated erbb2 , rac and pak ( hansen and linthicum , 2004 ; kaempchen et al . 2003 ; utermark et al ., 2003 ). loss of sch expression in scs could allow unrestricted autocrine stimulation of erbb2 / erbb3 , resulting in increased β1 integrin levels and prolonged activation of pka , rac - pak , ras - erk and pi3k / akt pathways . activation of these signaling cascades would stimulate a slow , but continuous proliferation of scs , characteristic of schwannoma growth in individuals with nf2 . in summary , we have shown that activation of erbb2 / erbb3 and β1 integrin receptors promotes phosphorylation of sch through distinct pka - and pak - dependent pathways . in vivo , these signaling cascades would cooperate to promote sc proliferation in response to axonal nrg and basal lamina adhesion . in its phosphorylated state , sch would also permit rac - pak dependent changes in the actin cytoskeleton associated with extension of processes along axons , a critical function for myelination . our findings shed light on sch &# 39 ; s function during development and pathogenesis in the peripheral nervous system . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . adam l , vadlamudi r , kondapaka s b , chernoff j , mendelsohn j , kumar r . 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