a common characteristic of these enzymes is that they are normally tightly regulated in unstimulated cells . stimulation ( e.g. , by binding of a growth factor to the extracellular domain of a receptor tyrosine kinase ) leads to a rapid , transient increase in tyrosine kinase activity . most oncogenic tyrosine kinases contain activating mutations and are dominant at the cellular level [ 2 , 3 ] . the human insulin - like growth factor 1 receptor ( igf1r ) is a heterotetramer containing two extracellular alpha subunits and two transmembrane beta subunits . binding of the ligand ( igf1 ) to the alpha subunits triggers a conformational change that leads to autophosphorylation of the intracellular kinase domains in the beta subunits . the signal is propagated through the pi 3-kinase and map kinase pathways to promote proliferation and cell survival . in the unstimulated state , the basal activity of the igf1r receptor is suppressed by autoinhibitory interactions between the activation loop and other residues in the kinase domain [ 68 ] and between the kinase domain and the juxtamembrane region . studies in cell culture systems have shown that overexpression of igf1r can lead to morphological transformation , while interference with igf1r reverses the transformed phenotype . igf1r is overexpressed in numerous solid tumors as well as in multiple myeloma [ 11 , 12 ] . the cell survival function of igf1r appears to be critical in these tumors , as inhibition of igf1r can induce apoptosis . a number of therapeutic approaches are currently being explored to interfere with igf1r signaling in cancer cells , including rna interference , receptor antibodies , and small molecule kinase inhibitors [ 11 , 12 ] . increased transcription of the igf1r gene has been shown to result from loss of tumor suppressor genes , such as , p53 or from the action of other oncogenes . loss of imprinting ( loi ) of igf2 is an epigenetic alteration found in many colorectal and other tumors . to date , no activating igf1r mutations have been identified in cancers . recent gene sequencing efforts have catalogued hundreds of somatic mutations in the coding regions of potential cancer genes . these mutations comprise both  driver  mutations ( which confer a growth advantage and are causally connected to the development of cancer ) and  passenger mutations , which do not contribute to the development of cancer . screening for somatic mutations in kinase genes identified two mutations in the gene encoding igf1r that led to aminoacid changes : a1347v ( from lung squamous cell carcinoma ) and an in - frame deletion of s1278 ( from renal clear cell carcinoma ) . the effect of these mutations , if any , on the biological function of igf1r has not been tested . m1255 falls in the c - terminal lobe of the tyrosine kinase catalytic domain , while s1278 and a1347 lie in the c - terminal portion of the receptor . we report the effects of the mutations on the in vitro biochemical activity of igf1r , as well as on the major igf1r signaling pathways in mammalian cells . mutant forms of igf1r were generated by site - directed mutagenesis ( quikchange kit , stratagene ) on the expression vector pbpv - igf1r . one million r - cells were plated onto 10  cm tissue culture dishes and grown to 50% confluency in dmem plus 4500  mg / l glucose ( fisher / cellgro ) , 10% heat inactivated fetal bovine serum ( vwr ) , 1x antibiotic / antimycotic ( fisher / cellgro ) and 50  ug / ml g418 ( sigma ) . r - cells were transfected with the pbpv - igf1r constructs using transit polyamine transfection reagent ( mirus ) according to the manufacturer 's instructions . after 24 hours , the transfection mixture was replaced with starvation media containing dmem , 1000  mg / l glucose ( invitrogen ) , 1x antibiotic / antimycotic ( fisher / cellgro ) , 0.5% bsa , 50  ug / ml g418 , and 50  ug / ml holotransferrin ( sigma ) . after 16 hours of starvation at 37c and 5% co2 , the cells were stimulated for 10  minutes at 37c with 40  ng / ml igf1 ( calbiochem ) . the cells were washed with 4c pbs ( sigma ) and lysed in 1  ml of lysis buffer containing 25  mm tris - hcl ( ph 8) , 2  mm edta , 140  mm nacl , 1% np40 , 0.5  ug / ml leupeptin , 0.5  ug / ml aprotinin , and 2  mm activated sodium orthovanadate . after clearing the lysates by centrifugation , 2  g of anti - igf1r antibody ( clone jbw902 , millipore ) was added to the lysates for 1  hour at 4c , followed by the addition of 60  l protein - a - conjugated agarose beads ( sigma ) . the precipitated proteins were analyzed on 10% sds polyacrylamide gels and transferred to pvdf membranes ( millipore ) . the blots were probed with anti igf1r antibody ( millipore ) , then stripped and reprobed with anti - igf1r ( pypy1135/1136 ) ( biosource ) . the blots were visualized using the supersignal west femto maximum sensitivity substrate system ( pierce ) . densitometry and analysis were performed using imagej version 1.42 and prism version 4 . in a separate set of experiments , the irs-1 receptor was immunoprecipitated in a similar manner using anti irs-1 ( millipore ) . cells were stimulated with 10  ng / ml igf1 ( the lower concentration was used to eliminate any contribution from insulin receptor ) . the blots were probed with antiphosphotyrosine ( 4g10 ) antibody . to analyze akt and map kinase activation , 50  ug of lysates was analyzed on 10% sds polyacrylamide gels , transferred to pvdf membranes and probed with antiphospho - akt and antiphospho - map kinase antibodies . membranes were stripped and reprobed with akt and map kinase antibodies for normalization ( cell signaling ) . to analyze the association of igf1r and rack1 , lysates ( 2  mg ) were immunoprecipitated with 4  g of anti igf1r c20 antibody ( santa cruz ) and 60  l protein - a - conjugated agarose beads . after analysis on 10% sds polyacrylamide gels and transfer to pvdf membranes , the blots were probed with anti rack1 ( bd transduction ) and anti igf1r ( clone jbw902 ) antibodies . dna sequences encoding igf1r residues 9301337 ( the complete cytoplasmic domain of igf1r ) had previously been subcloned into pfastbac htb ( life technologies , inc . ) . site directed mutagenesis was performed to generate the m1255i , s1278 , and a1347v mutants . after confirming the mutations by dna sequencing , recombinant virus was generated using the bac - to - bac system ( life technologies , inc . ) . the virus was then used to produce protein by infection of spodoptera frugiperda ( sf9 ) cells . one billion sf9 cells were infected with high - titer virus and harvested after 72  hours . the cells were lysed in a french pressure cell in lysis buffer containing 50  mm tris - hcl ( ph 8.5 ) , 5  mm edta , 1% triton - x 100 , 5  mm 2-mercaptoethanol , 0.5  ug / ml aprotinin , 0.5  ug / ml leupeptin , 2  mm pmsf , and 2  mm activated sodium orthovanadate . the clarified lysate was loaded onto a 5  ml column of ni - nta resin ( qiagen ) which had been preequilibrated with buffer a ( 20  mm tris - hcl ph 8.5 , 500  mm kcl , 10% glycerol ) . imidazole ( 20  mm ) and 2-mercaptoethanol ( 5  mm ) were added to buffer a after the column had been equilibrated . the column was washed with 10 column volumes of buffer a containing imidazole ( 20  mm ) and 2-mercaptoethanol ( 5  mm ) , followed by 2 column volumes of buffer b ( 20  mm tris - hcl ph 8.5 , 1  m kcl , 5  mm 2-mercaptoethanol , 10% glycerol ) , then 10 column volumes of buffer a. the igf1r cytoplasmic domain was eluted with buffer c ( 20  mm tris - hcl ph 8.5 , 100  mm kcl , 100  mm imidazole , 5  mm 2-mercaptoethanol , 10% glycerol ) . the fractions were analyzed on 10% sds - polyacrylamide gels , and the peak fractions were pooled and stored in aliquots at 80c in 40% glycerol . the experiments were performed at 30c in buffer containing 100  mm tris - hcl ( ph 7.5 ) , 10  mm mgcl2 , 1.5  mm phosphoenolpyruvate , 201  u / ml pyruvate kinase , 230  u / ml lactate dehydrogenase , and 0.5  mm nadh . the concentration of the enzyme was 2  m and the concentration of atp was 1  mm . for peptide phosphorylation experiments , the enzymes were prephosphorylated by incubation with 5  mm atp for 15  minutes at room temperature prior to the assays . the final concentration of the enzymes in these assays was 800  nm , the concentration of the atp was 1  mm , and the concentration of the peptide was 1  mm . we expressed full - length igf1r harboring the m1255i , s1278 , or a1347v mutations in fibroblasts derived from igf1r - deficient mice ( r - cells ) . for comparison as an initial check of the activity of the receptors , we measured autophosphorylation at y1135/y1136 . tris - phosphorylation of igf1r at y1131/y1135/y1136 in the activation loop produces a large increase in kinase activity . we immunoprecipitated igf1r from the r - cells , separated proteins by sds - page and analyzed them by western blotting with a phospho - specific antibody to py1135/py1136 ( figure 1 ) . membranes were stripped and reprobed with an anti - igf1r antibody ; normalization of the py1135/py1136 signal with respect to total igf1r revealed no significant change in levels of autophosphorylation . thus , the mutations did not drastically affect the intrinsic tyrosine kinase activity of igf1r . we previously reported the expression and purification of the entire cytoplasmic portion of igf1r ( i.e. , juxtamembrane , kinase , and c - terminal domains ) from insect cells using a baculovirus expression vector . we generated recombinant baculoviruses encoding the m1255i , s1278 , and a1347v mutant forms of igf1r . we infected spodoptera frugiperda cells with mutant or wild - type igf1r baculoviruses , harvested cells after three days of infection , and purified the proteins ( supplemental figure 1 ) supplementary materials are available online at doi:10.1155/2012/804801 . all forms of igf1r tested were active in the in vitro autophosphorylation assay ( figure 2 ) . as observed previously for wild - type igf1r , the enzyme progress curves were biphasic , due to the fact that autophosphorylation increases the enzymatic rate . the rates of wild - type and a1347v igf1r were comparable , while the rates of the s1278 and m1255i mutant forms were somewhat slower . next , we examined the ability of the purified receptors to phosphorylate an exogenous substrate . in these experiments , we used a synthetic peptide substrate previously shown to be phosphorylated by igf1r with good kinetic parameters [ 6 , 20 ] . to eliminate the effect of autophosphorylation , we first prephosphorylated the proteins by treatment with atp [ 9 , 21 ] . the wild - type and mutant forms of igf1r were all active in this assay ( figure 3 ) . the m1255i mutation , which occurs toward the c - terminal end of the kinase catalytic domain , showed a small reduction in peptide phosphorylation . taken together , the receptor phosphorylation assays ( figure 1 ) and the autophosphorylation and peptide phosphorylation assays ( figures 2 and 3 ) showed that the mutant kinase domains were active but were not hyperactivated relative to wild type . the insulin receptor substrate ( irs ) family adaptor proteins are important proximal substrates for the activated insulin and igf1 receptors [ 22 , 23 ] . irs-1 contains n - terminal ph and ptb domains which play important roles in directing the adaptor protein to ir / igf1r . these phosphorylated sequences provide binding sites for grb2 , phosphoinositide 3-kinase , nck , shp2 , and other cellular signaling proteins containing sh2 domains . we expressed wild - type and mutant forms of igf1r in r - cells and analyzed the overall tyrosine phosphorylation of endogenous irs-1 by immunoprecipitation and western blotting with anti - ptyr antibodies . these results showed that the mutant forms of igf1r were capable of phosphorylating irs-1 ( figure 4 ) . after transfection with igf1r expression plasmids , r - cells were serum starved , then stimulated with igf1 or left unstimulated . we measured akt activation in cell lysates using an activation state - specific antibody ( figure 5 ) . figure 5 shows a representative western blot , along with a bar graph depicting a summary of all the experiments . akt activation in the presence of igf1 was similar for the various forms of igf1r tested ( figure 5 ) . the basal , unstimulated levels of akt activity were 1.52-fold higher for the s1278 , a1347v , and m1255i forms of igf1r , and the fold increases ( stimulated / unstimulated ) were consequently lower . as an attempt to gain insight into the basis for increased levels of phospho - akt in the absence of igf1 , we analyzed the association between igf1r and the rack1 adaptor protein . the association between igf1r and rack1 is independent of receptor activation , but a ser - to - ala mutation at s1278 of igf1r was reported to block rack1 binding . because one of the cancer - associated mutations involved the same residue ( s1278 ) , we measured the coimmunoprecipitation of endogenous rack1 with wild - type and mutant forms of igf1r in r - minus cells . association with rack1 was unaffected by the s1278 and other mutations ( figure 6 ) . next , we carried out similar experiments to measure activation of map kinase in r - cells expressing wild - type or mutant forms of igf1r . mapk activity was measured by western blotting with a phosphospecific antibody , and total mapk levels were assessed by reprobing with a mapk antibody . as expected , igf1 stimulation led to an increase in map kinase activation in r - cells expressing wild - type igf1r . hormone stimulation of r - cells expressing mutant forms of igf1r led to levels of map kinase activation that were not significantly different from wild type . on the other hand , the basal ( unstimulated ) levels of map kinase activation were 2.5 - 3-fold higher for the mutants that for wild type ( figure 7 ) . thus , the fold increases ( stimulated / unstimulated ) for the m1255i , s1278 , and a1347v mutants were 2.1 , 2.0 , and 2.8 , respectively , compared to 4.8 for wild type . these results , and the similar findings for akt signaling ( figure 5 ) , suggest that the regulatory interactions that normally keep igf1r activity in check were partially disrupted in the case of the cancer - associated mutants . for several receptor tyrosine kinases , biochemical and structural studies have implicated the c - terminal portion of the cytoplasmic domain of several rtks in kinase signaling . the deletion of the c - terminal residues of erbb2 increased receptor kinase activity as well as transforming ability . a peptide containing the c - terminus of pdgfr  receptor inhibited its kinase activity . the crystal structure of the tie2 rtk has been solved with an intact c - terminal tail . the tie2 c - terminal tail interacts with the c - lobe of the kinase catalytic domain and ends near the substrate binding site . deletion of the tie2 c - terminal tail increased receptor autophosphorylation and kinase activity , lending support to a model in which the tail is involved in kinase regulation . current evidence suggests that the c - termini of ir and igf1r do not play a direct role in controlling the activation kinetics of the catalytic domains [ 30 , 31 ] . on the other hand , a chimeric igf1r containing the c - terminal domain of ir was more potent in stimulating glycogen synthesis than wild - type igf1r , suggesting that biological specificity resides ( at least in part ) in the c - terminus . this idea has been reinforced by studies demonstrating differential binding and phosphorylation of cellular proteins mediated by the c - termini of igf1r and ir . ceacam1 is an example of a protein that binds specifically to the c - terminus of ir , 14 - 3 - 3 proteins and iip-1 bind selectively to the igf1r c - terminus [ 34 , 35 ] , and rack1 interacts with both c - termini . protein - protein interactions mediated by the c - terminus play a role in downstream igf1r signaling . in particular , deletion of the igf1r c - terminus increased the ability of the receptor to promote cell survival , suggesting that the c - terminus has a negative regulatory role . consistent with this idea , tumor cell apoptosis was increased by stable expression of a myristoylated igf1r c - terminus   or by introduction of a synthetic 17-aminoacid peptide from the c - terminus . point mutations in the igf1r c - terminus inhibit cell survival   and anchorage - independent growth , while deletion of the c - terminus blocks receptor ubiquitination . two of the cancer - associated igf1r mutations studied in this paper ( s1278 and a1347v ) fall in the c - terminus and one ( m1255i ) is in the c - terminal lobe of the catalytic domain . consistent with prior mutational analyses of the igf1r c - terminus , the cancer - associated mutations did not significantly affect full - length receptor autophosphorylation in cells ( figure 1 ) . similarly , the purified cytoplasmic domains retained their autophosphorylation activity in vitro and their ability to phosphorylate exogenous peptide substrates ( figures 2 and 3 ) . phosphorylation of the receptor - proximal substrate irs-1 also appeared to be unaffected by the mutations ( figure 4 ) . for comparison , a mutagenic analysis of the igf1r / ir juxtamembrane regions showed that these residues exert direct negative control over receptor phosphorylation and activity . because previous studies suggested both positive and negative roles of the igf1r c - terminus in cell signaling , it was not clear what the predicted effects of the cancer - associated mutations would be . while the mutant forms of igf1r showed no difference from wild - type with respect to the maximal levels of map kinase and akt activation , basal levels of activity were higher ( figures 5 and 7 ) . these results are consistent with a model in which the igf1r c - terminus plays an inhibitory role in signal transduction , and the mutations partially destabilize the inhibited conformation , leading to enhanced activity . although the effects observed were modest , these mutations represent the first activating mutations of igf1r identified in human cancers . the mutations studied here do not fall within the sequence ( 13121328 ) included in the inhibitory synthetic peptide which was previously shown to promote tumor cell apoptosis . the alterations in map kinase and akt signaling are likely due to changes in protein - protein interactions with the igf1r c - terminus . in principle , the mutations might increase binding of a positive mediator of igf1r signaling or block binding of an inhibitory protein factor . rack1 binding requires s1278 in the igf1r c - terminus , since a ser - to - ala mutation eliminates binding . although one of the cancer - associated mutations is a deletion of this same residue , rack1 binding was unaffected in our experiments . future experiments will be aimed at identifying igf1r - interacting partners that are altered in the cancer - associated variants .