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Cell Growth & Differentiation Vol. 11, 305-314, June 2000
© 2000 American Association for Cancer Research

Src Family Kinase Activity Is Required for Kit-mediated Mitogen-activated Protein (MAP) Kinase Activation, However Loss of Functional Retinoblastoma Protein Makes MAP Kinase Activation Unnecessary for Growth of Small Cell Lung Cancer Cells1

Cornelius Bondzi, Julie Litz, Paul Dent and Geoffrey W. Krystal2

Departments of Microbiology/Immunology [C. B., G. W. K.], Medicine [J. L., G. W. K.], and Radiation Oncology [P. D.], Medical College of Virginia of Virginia Commonwealth University and McGuire Veterans Affairs Medical Center, Richmond, Virginia 23249


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Numerous characteristic genetic alterations have been described in SCLC.3 These include almost universal loss of functional p53 and Rb, amplification or overexpression of a Myc family member, overexpression of Bcl-2, and loss of an as-yet-unidentified tumor suppressor gene located on chromosome 3p (1, 2, 3) . In addition, multiple autocrine loops play an important role in the growth factor independence of this tumor. Autocrine loops involving gastrin-releasing peptide (4) , IGF-1 (5) , and SCF have been described. Coexpression of SCF and its receptor, Kit, occurs in over 70% of cell lines and primary tumors (6, 7, 8) , and SCF can enhance the proliferation and motility of selected cell lines (9 , 10) . The reconstruction of this autocrine loop in a rare cell line that expressed only SCF resulted in enhanced growth-factor independence, and the inhibition of Kit function by using a DN receptor or small-molecule inhibitors of Kit kinase activity resulted in the growth inhibition of multiple cell lines that coexpressed SCF and Kit (10 , 11) .

The c-kit gene encodes a transmembrane tyrosine kinase highly related to the receptors for PDGF and CSF-1 (12) . Whereas Kit is capable of activating multiple signal transduction pathways (13) , the pathways responsible for the proliferation of SCLC cells have not been defined. Evidence obtained using microinjection of inhibitory antibodies or DN expression constructs indicates that both the PDGFR and the CSF-1R require active Src family kinases to initiate DNA synthesis in fibroblasts (14 , 15) . However, using the alternative approach of mutating the Src binding sites on the PDGFR, other investigators have concluded that activation of Src kinases is not necessary for PDGF-induced proliferation (16, 17, 18) . We hypothesized that activation of Src family kinases could be involved in SCF-mediated proliferation of SCLC based on the fact that ectopic expression of Lck occurs in some SCLC cell lines (19) that also express high levels of Kit and on the observation that Src kinases are activated in SCLC (20) . We recently demonstrated that, in the H526 cell line, the proliferation of which can be stimulated by exogenous SCF, SCF induced both the physical association of Lck and Kit as well as an activation of Lck that was equivalent to that seen after the stimulation of T cells (21) . The time course of Lck activation paralleled that of MAPK activation, which suggested that Lck activation plays a role in early events in Kit signal transduction; the present study explores that role.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Kit-mediated MAPK Activation Is Inhibited by PP1.
Previous studies had demonstrated that activation of Lck and ERK 1 occurred with similar kinetics after treatment of H526 cells with SCF, with activation peaking 5 min after the addition of SCF (21) . To determine whether ERK 1/2 activation was linked to Lck activation, H526 cells were stimulated with SCF for 5 min in the presence of the Src family kinase inhibitor PP1 (22) or of DMSO vehicle and assayed for ERK 1/2 activation by Western blotting with an activation-specific antibody. Previous studies (21) demonstrated that a 30-min in vivo pretreatment with 10 µM PP1 blocked Lck activity by at least 90% using an immune complex kinase assay, without having a significant effect on Kit kinase activity. SCF induced an approximately 10-fold increase in the amount of activated ERK 1 and 2 over nonstimulated cells in this assay; this increase was completely blocked by the presence of PP1 (Fig. 1A)Citation . As a control, H526 cells were also treated with IGF-1 because it is an excellent mitogen for these cells and PP1 had little effect on IGF-1-stimulated proliferation (21) . IGF-1 induced an approximately 2-fold increase in active MAPK (predominantly ERK 2), which was unaffected by the presence of PP1. These results were confirmed using a myelin basic protein phosphorylation assay (Fig. 1B)Citation . Thus, PP1 specifically inhibited SCF-mediated activation of ERK 1 and 2; IGF-1-mediated ERK activation was not affected by PP1.



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Fig. 1. PP1 blocks SCF-stimulated MAPK activation. A, H526 cells incubated in serum-free medium overnight were either left untreated (NS), or stimulated with 100 ng/ml SCF or 20 ng/ml IGF-1 in the presence of DMSO vehicle or 10 µM PP1 and lysed in SDS sample buffer after 5 min. Duplicate samples were resolved by PAGE through a 10% gel, Western blotted, and stained with either an activation-specific or a pan-ERK antibody. SCF stimulation resulted in a 10-fold increase in MAPK activity (determined by densitometry), which was completely inhibited by PP1. IGF-1 resulted in a 2-fold increase in MAPK activity that was not affected by PP1. B, H526 cells were treated as above, lysed, and ERK 1 and 2 were immunoprecipitated with a pan-ERK antibody. The immunoprecipitate was incubated in the presence of myelin basic protein and {gamma}32P-ATP in triplicate and the labeled myelin basic protein was bound to phosphocellulose paper, was washed, and was quantitated by scintillation counting.

 
These results suggested that activation of the Src family kinases is required in order for Kit to activate MAPK. This conclusion, however, hinges on the results of the immune complex kinase assay (21) accurately predicting that Kit enzymatic activity is not affected by PP1. Because the initial study characterizing PP1 did not test its in vitro activity against PDGFR class receptors (22) , we decided to confirm the immune complex kinase assay by determining the effect of direct addition of PP1 to an in vitro kinase assay (Fig. 2)Citation . H526 cells were stimulated with SCF and Kit was immunoprecipitated. Preincubation of the cells in 10 µM PP1, which blocked Lck phosphorylation by 90% (21) , had little effect on Kit phosphorylation (Fig. 2, Lane BCitation ), as previously reported. However, the addition of 10 µM PP1 directly to the kinase assay eliminated Kit autophosphorylation in vitro (Fig. 2, Lane CCitation ). To confirm the in vitro results, H526 cells were stimulated with SCF in the presence of an increasing concentration of PP1; Kit was then immunoprecipitated and assayed for phosphotyrosine content. PP1 efficiently inhibited SCF-stimulated Kit tyrosine phosphorylation with an IC50 between 0.5 and 1 µM (Fig. 3A)Citation ; 10 µM PP1 inhibited Kit tyrosine phosphorylation 10- to 20-fold. The magnitude of this effect at higher PP1 concentrations suggests that the observed results were not merely an inhibition of Lck-mediated tyrosine phosphorylation of the receptor. Taken together, these data demonstrate that PP1 inhibits Kit kinase activity but that this inhibition can be reversed during the IP steps of an immune complex kinase assay.



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Fig. 2. PP1 directly inhibits Kit in an IP-kinase assay. H526 cells were stimulated with SCF using cells pretreated with DMSO vehicle (Lanes A and C) or 10 µM PP1 (Lane B) for 30 min. A Kit IP was performed, followed by an in vitro kinase assay using radiolabeled ATP. PP1 (10 µM) was added to one reaction (Lane C) before the addition of ATP; an equivalent volume of DMSO was added to the others. The kinase reaction was electrophoretically resolved and transferred to nitrocellulose, and an autoradiograph was performed (upper panel), followed by staining for total Kit protein (lower panel). The addition of PP1 directly to the kinase reaction inhibited Kit autophosphorylation in vitro, whereas the addition of PP1 to cells prior to lysis had little effect on in vitro kinase activity.

 


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Fig. 3. PP1 blocks both SCF-mediated Kit and MAPK activation. In A, H526 cells incubated in serum-free medium overnight were either left unstimulated or stimulated for 5 min with 100 ng/ml of SCF after a 30-min pretreatment with the indicated concentrations of PP1. Kit was immunoprecipitated and the immunoprecipitate was immunoblotted with antiphosphotyrosine and anti-Kit antibodies in succession. The degree of Kit activation was calculated by densitometry (ratio of pTyr:Kit signal) and normalized to the SCF-stimulated vehicle control lane which was arbitrarily assigned a value of 100%. Experiment shown is representative of three replicates. In B, cells were treated as above, but after SCF treatment, whole-cell lysates were made in SDS buffer. The lysates were immunoblotted and stained successively with antiphospho-ERK and antipan-ERK antibodies. The degree of activation was calculated as above. Experiment shown is representative of three replicates.

 
To determine whether the inhibition of MAPK correlated more closely with the sensitivity of Kit to PP1 or the reported sensitivity of Lck to the drug (in vitro and cellular IC50s of 5–20 nM and 0.5 µM, respectively; Ref. 22 ), we performed a dose-response analysis while assaying for MAPK activation. Fig. 3BCitation illustrates that SCF-mediated MAPK activation was more sensitive to PP1 than SCF-mediated Kit activation. Concentrations of PP1 in the 1–10 µM range reproducibly brought MAPK activity to basal levels, with significant residual Kit activity still present at the same concentrations. However, because the sensitivities of MAPK and Kit to PP1 were overlapping, we could not draw any firm conclusions as to the involvement of Lck based on the differential sensitivity to PP1. However, we reasoned that if the greater sensitivity of MAPK activation was caused by the involvement of Lck, then overexpression of Lck could decrease the sensitivity of MAPK activation to PP1. We, therefore, compared the sensitivity of SCF-mediated MAPK activation to PP1 in a cell line engineered to overexpress murine Lck under the control of a doxycycline-inducible promoter with that of the same cell line in the absence of doxycycline and that of a control cell line transfected with empty vector. Fig. 4Citation illustrates that, in both the empty vector and the uninduced controls, MAPK activity was inhibited to or below basal levels (seen in the absence of SCF) by 1–10 µM PP1. Although PP1 in the same concentration range also inhibited MAPK activity in cells overexpressing Lck, activity remained well above basal levels, which demonstrated that overexpression of Lck can induce a partial resistance to PP1. The ability of PP1 to inhibit Kit autophosphorylation was not altered by Lck overexpression. These results support the involvement of Lck in SCF-mediated MAPK activation.



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Fig. 4. Overexpression of Lck decreases the sensitivity of SCF-mediated MAPK activation to PP1. H526 cells were stably transfected with the pTet-On plasmid followed by the pTRE plasmid containing either no insert (empty vector) or the murine WT Lck cDNA. Cultures of cells containing pTRE and the WT Lck expression plasmid were treated with doxycycline for 24 h; a second culture of WT Lck-containing cells was not induced with doxycycline. All of the cultures were incubated in serum-free media overnight and were left unstimulated or were stimulated for 5 min with 100 ng/ml SCF after a 30-min preincubation with the indicated concentrations of PP1. Each culture was split for making whole-cell lysates (for analysis of ERK activation and Lck expression) and for Kit IP. Far-right column, the antibodies that were used to stain each immunoblot. Whole-cell lysates were initially stained for active ERK, were stripped, and were then stained with the pan-ERK antibody. Immunoprecipitates were first stained for phosphotyrosine followed by Kit. Only the Kit IP from the induced WT Lck culture is shown; others showed a pattern of Kit inhibition similar to that illustrated here and in Fig. 3Citation . Far-left column, the relative levels of Lck expression in each culture. Persistence of ERK activation above basal levels (levels in cells not treated with SCF) in the PP1-treated WT Lck + Dox culture indicates that Lck overexpression induces partial resistance to PP1. Densitometric analysis revealed the following normalized percentage of ERK activity in cells treated with SCF in the presence of 10 µM PP1 relative to basal ERK activity (last lane ÷ first lane x 100): pTRE + Dox, 61%; WT Lck - Dox, 70%; WT Lck + Dox, 257%. Dox, doxycycline.

 
DN Lck Blocks Kit-mediated MAPK Activation.
To confirm the involvement of Lck in SCF-mediated MAPK activation, H526 subclones capable of inducible expression of a DN form of Lck were derived. A mutation that completely inactivates the kinase activity, K273R (23) , was introduced into the murine Lck cDNA. In addition, a second mutation, Y505F, was also introduced. The latter mutation prevents phosphorylation of Y505 by Csk kinases, which, in turn, would prevent its binding to the SH2 domain (24) . This should theoretically result in a more potent DN form by allowing freer access to the SH2 and SH3 domains for proteins that interact with Lck. By itself, the Y505F mutation results in constitutively enhanced enzymatic activity (24) . The WT DN Lck cDNA, and a Lck cDNA containing the Y505F mutation alone, were cloned into the pTRE vector that contains seven tandem copies of the Tet operator sequence upstream of the minimal human cytomegalovirus promoter. These expression constructs and the empty vector were transfected into the H526-IC4 subclone, which was made to constitutively express the "reverse" Tet repressor protein (25) , which allowed doxycycline-inducible expression. Subclones that demonstrated doxycycline-inducible expression of the heterologous Lck proteins were selected, and the ability of SCF to induce ERK 1 and 2 activation before and after doxycycline induction was assessed by Western blotting with the activation-specific antibody. Fig. 5Citation illustrates that the expression of WT and Y505F Lck had no effect on SCF-mediated MAPK activation, whereas expression of the DN mutant efficiently blocked MAPK activation. This inhibition seemed to be dose dependent, because the basal expression of DN Lck in the absence of doxycycline reproducibly had a modest effect on MAPK activity and doxycycline treatment markedly enhanced the effect. Importantly, the Y505F mutant did not induce MAPK activation in the absence of SCF, which suggested that the concerted effects of both Kit and Lck are necessary for MAPK activation. Thus, experiments using expression of a DN mutant Lck confirm the differential sensitivity of SCF-mediated MAPK inhibition to PP1. Taken together, these data indicate a requirement for Src family kinases, and in particular, Lck in the H526 cell line, in the signal transduction pathway leading from Kit to ERK activation.



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Fig. 5. DN Lck blocks SCF-mediated MAPK activation. H526 cells were stably transfected with the pTet-On plasmid followed by the pTRE plasmid containing either no insert (empty vector), the WT Lck cDNA, the Y505F Lck cDNA containing the constitutively activating mutation, or the DN Lck cDNA containing both the Y505F and K273R mutations. SCF-stimulated MAPK activation was assessed by immunoblotting with the activation-specific antibody in the presence or absence of doxycycline-induced expression of WT and mutant Lck. The extent of MAPK activation can also be estimated by assessing the mobility shift seen after staining with the pan-ERK antibody. A and B illustrate two representative experiments using two independently derived DN clones. In all, three independently derived clones expressing DN Lck were assayed in duplicate with consistent results. A, arrow, a background band intermittently detected between ERK 1 and ERK 2; the intensity of this band is independent of doxycycline or SCF treatment.

 
ERK Activation Is Not Required for SCF-mediated Growth.
Although the data above clearly implicate Src family kinases in the activation of the ERKs, they do not address whether ERK activation is necessary for SCF-stimulated proliferation. This appeared to be a crucial question, in light of the fact that the extent of ERK activation did not correlate well with the mitogenicity of different growth factors. IGF-1 is consistently a better mitogen for H526 cells than is SCF (21) , yet the degree of ERK activation by SCF is consistently much greater than that produced by IGF-1 (Fig. 1)Citation . To initially address this question, the growth of H526 cells expressing DN Lck were compared with cells overexpressing the WT protein or control cells containing the empty vector. Fig. 6Citation illustrates that expression of DN Lck, which represses SCF-mediated MAPK activation, had no effect on cell growth, which suggests that MAPK activation is not necessary for SCF-mediated growth.



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Fig. 6. DN Lck does not inhibit SCF-stimulated growth. H526-IC4 cells containing the "reverse" Tet repressor and the empty pTRE vector or vectors encoding WT Lck or DN Lck were induced with doxycycline for 24 h, made quiescent in serum-free medium (containing doxycycline) overnight, and then left either unstimulated () or stimulated with 100 ng/ml SCF ({blacksquare}). Growth was assessed by MTT assay after 72 h. Error bars, the SD from the mean of eight replicate wells; results are representative of three replicate experiments.

 
Because the inhibition of Src kinases could have pleiotropic effects, we sought to confirm the above observations by directly inhibiting the MAPK pathway. ERK activation after SCF treatment was inhibited using the MEK inhibitor PD 98059 (26) . As shown in Fig. 7Citation , 40 µM PD 98059 completely inhibited SCF-mediated MAPK activation, yet concentrations of the inhibitor up to 100 µM had no effect on SCF-mediated growth. This strongly suggests that although Src family kinases are required for SCF-mediated ERK activation, SCF must also activate one or more alternative signal transduction pathways that are necessary and sufficient for transducing the growth stimulatory signal. In addition, we also observed that PD 98059 at concentrations up to 100 µM had no significant effect on the serum-stimulated growth of H526 and five additional SCLC cell lines (not shown), which suggested that MAPK activation is also not necessary for serum-stimulated growth.



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Fig. 7. Inhibition of MAPK activation fails to block SCF-mediated growth. In A, H526 cells were pretreated with 40 µM PD 98059 or DMSO vehicle for 30 min and then left either unstimulated (NS) or stimulated with SCF (100 ng/ml) for 5 min. MAPK activation was assessed by Western blotting. In B, H526 cells in serum-free medium containing SCF were incubated in increasing concentrations of PD 98059 and growth over 72 h was measured using the MTT method. Despite efficiently inhibiting MAPK activation, the MEK inhibitor had no effect on growth. Error bars, the SD from the mean of eight replicate wells; results are representative of three replicate experiments.

 
Expression of Rb Restores Sensitivity to MAPK Inhibition.
We were quite surprised that inhibition of ERK activation had no demonstrable effect on growth, despite the documented importance of this pathway to the proliferation of a wide range of cell types (27) . We reasoned that one possible explanation for this result was that genetic alterations inherent in SCLC eliminated the requirement for ERK activation. One clear candidate for this genetic alteration was the loss of functional Rb. Activation of cyclin D expression has been shown to be a direct consequence of MAPK activation in rodent fibroblasts (28) , and, thus, it seemed the that loss of Rb could eliminate the need for this consequence of MAPK activation. To test this hypothesis, H526-IC4 cells containing the doxycycline-responsive transcriptional activator were transfected with the empty pTRE vector or the vector containing the WT Rb cDNA. The expression pattern of several stable subclones is illustrated in Fig. 8ACitation . The ability of PD 98059 to block cells that contain empty vector or that express Rb from entering S phase after SCF stimulation was tested using [3 H]thymidine incorporation. Relative to controls incubated in the absence of the MEK inhibitor, DNA synthesis by Rb-expressing subclones was inhibited by 25–60%, whereas cells containing empty vector were unaffected (Fig. 8B)Citation . Thus, it appears that expression of Rb can partially restore the ability of the MEK inhibitor to block proliferation. The inability of PD 98059 to completely block DNA synthesis in the presence of Rb expression may be attributable to the high levels of N-Myc expression the H526 cell line displays secondary to N-myc amplification (29) ; overexpression of N-Myc is known to abrogate the G1-S phase checkpoint (30) . It is possible that additional genetic alterations are also partly responsible for the resistance of SCLC cells to inhibition of MAPK activation.



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Fig. 8. Expression of Rb partially restores sensitivity to PD 98059. In A, H526-IC4 cells containing the "reverse" Tet repressor were transfected with the empty pTRE plasmid or with the pTRE plasmid containing the Rb cDNA. Individual subclones were isolated and analyzed for Rb expression in the absence and presence of doxycycline by Western blotting; shown is the Mr 110,000 region of the blot. In B, subclones expressing Rb or containing empty vector were cultured in serum-containing medium with doxycycline for 24 h to induce Rb expression, were serum-starved for 6 h, and then were placed in serum-free medium containing 100 ng/ml SCF in the presence of DMSO vehicle (control) or 80 µM PD 98059. The subclones were incubated for 12 h, at which time 1 µCi/ml [3H]thymidine was added, and the incubation was continued for an additional 3 h. The cells were then collected and [3H]thymidine incorporation was determined by scintillation counting. The data are expressed as the percent of thymidine incorporation by cultures containing PD 98059 relative to cultures containing vehicle; the data are representative of four independent experiments.

 
To document the observation that inhibition of DNA synthesis by PD 98059 correlated with hypophosphorylation of Rb, the Rb 10D5 subclone was incubated in [32P]Pi and then was stimulated with SCF in the presence or absence of drug. After a 12-h incubation, Rb was immunoprecipitated and electrophoretically resolved. Fig. 9Citation illustrates a decrease in the amount of highly phosphorylated Rb, distinguished by its slower mobility, in the PD 98059-treated cells, thus correlating the inhibition of ERK activation with Rb hypophosphorylation and the inhibition of DNA synthesis. Staining with phospho-specific antibodies revealed that the phosphorylation of specific serines was selectively affected by inhibition of the MAPK cascade. Whereas phosphorylation of serine 780 seemed to occur in the presence of the MEK inhibitor, phosphorylation of serine 795, which correlated with the presence of the slower mobility form of Rb, seemed to be almost completely blocked by PD 98059. Interestingly, phosphorylation of this residue has been correlated with relief of Rb-induced cell cycle arrest in microinjection assays (31) .



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Fig. 9. Inhibition of ERK activation lead to site-specific Rb hypophosphorylation. Rb expression was induced in the 10D5 subclone by a 24-h incubation in serum-containing medium with doxycycline followed by serum-starvation for 6 h. The cells were then placed in serum-free medium containing 100 ng/ml SCF and 1 mCi/ml [32P]Pi in the presence of DMSO or 80 µM PD 98059. The incubation was continued for 12 h, at which time Rb was immunoprecipitated, electrophoretically resolved, and analyzed by autoradiography and immunoblotting using antibodies directed against total Rb and phospho-specific isoforms. After probing for total Rb protein, the blot was stripped and stained with a pool of three antibodies directed against phospho-peptides containing pSer807/811, pSer780, and pSer795. The blot was then stripped and stained sequentially with each antibody individually (in the above order). The staining with the pSer807/811 antibody is not shown because it was completely negative. Rb phosphorylated on Ser795, present in the slower mobility band, is selectively lost after treatment with PD 98059.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have demonstrated that in the H526 SCLC cell line, Lck activity is required for SCF-stimulated MAPK activation using two complementary techniques: (a) although activation of both Kit and MAPK were sensitive to inhibition by the tyrosine kinase inhibitor PP1, MAPK inhibition occurred at a lower PP1 concentration; this greater sensitivity to PP1 could be partially reversed by overexpression of Lck; and (b) inducible expression of a kinase-inactive DN Lck protein blocked SCF-mediated MAPK activation in a dose-dependent fashion. Although these observations run contrary to the dogma that receptor tyrosine kinases activate the Ras-MAPK pathway directly, they are consistent with a large body of data that demonstrates the importance of Src kinases in signaling from class-III RTKs, including the PDGFR, CSF-1R, and Kit. In murine fibroblasts expressing the PDGFR or the CSF-1R, interaction with their respective ligands recruits Src family kinases to the activated receptors and results in an increase in the specific activity of Src (32 , 33) . The Src family members Lyn and Lck have also been shown to associate with, and become activated by, Kit in hematopoietic (34) and SCLC cells (21) . Microinjected anti-Src antibody or plasmids encoding a dominant inhibitory Src mutant blocked DNA synthesis mediated by PDGFR and CSF-1R stimulation, which suggests that Src is an obligatory participant in the PDGF and CSF-1 mitogenic pathways (15) . Both the SH2 and SH3 domains of Src kinases were necessary for participation in these signaling pathways (14 , 35 , 36) , which suggested that Src kinases must be able to interact with both the receptor and downstream signaling molecules. The precise role of Src kinases in these pathways has been elusive. Attempts to rescue cells from the effects of DN Src have shown that overexpression of Fos and Jun cannot, but overexpression of Myc can, overcome the inhibition of DNA synthesis, which suggests that activation of Src is linked to transcriptional activation of c-myc and entry into S phase (37) . Evidence also exists for a role for Src activation at the G2-M transition (38) .

Other groups have taken the alternative approach of mutating the juxtamembrane tyrosines of the class-III receptors required for Src binding rather than directly inhibiting Src kinases (16, 17, 18) . Although they universally have failed to define a role for Src kinases in proliferative signal transduction mediated by the PDGFR using this approach, cells expressing the mutant receptor did show decreased phosphorylation of multiple downstream SH2-containing signaling molecules and an inability to efficiently activate ERK 1 and 2 at high ligand concentrations (16) . This last observation is consistent with our findings. Timokhina et al. (39) took a similar approach, demonstrating that the Y567F mutation, which abolished Fyn binding to murine Kit, had no effect on SCF-mediated proliferation of bone marrow-derived mast cells. More recently Lennartsson et al. (40) have demonstrated that the homologous Y568F mutation in human Kit blocked SCF-mediated Src kinase and Ras/MAPK activation in transfected PAE cells, in excellent agreement with our findings. They localized the requirement for Src kinases to a step proximal to, or at the level of, Shc phosphorylation. Interestingly, they observed that the Y568F mutation did not significantly affect SCF-mediated proliferation, but the Y568F, Y570F double mutant resulted in a severely impaired proliferative response.

The inconsistencies between studies regarding the requirement for Src kinase activation for activation of MAPK and proliferation by class-III RTKs are likely attributable to numerous factors. Some discrepancies are likely to be attributed to potential functional differences between the individual receptors. Undoubtedly, the cellular background in which these receptors are expressed plays a role, as evidenced by our observation that the lack of a requirement for MAPK activation for proliferation is in part dependent on the loss of functional Rb in SCLC cells. Most importantly, however, the technique used to interfere with Src kinase activity likely affects the outcome and interpretation of the data. Whereas the juxtamembrane tyrosines of Kit are clearly important for binding Src kinases, they also constitute the binding sites for the SHP phosphatases (41) and the CHK kinase (42) , which suggests that their mutation could cause pleiotropic effects. On the other hand, microinjected antibodies or DN proteins could interfere with other critical Src functions unrelated to RTK signaling. It has been suggested that the discrepancy between studies as to the requirement for Src kinases to initiate proliferation by class-III RTKs may relate to the unperturbed basal level of Src activity in cells containing the mutant receptors (16) . Our studies cannot distinguish between a requirement for Src kinase activation versus basal Src kinase activity, because it is likely that both are affected by PP1 and DN Lck expression. It is important, however, to point out that activation of Lck alone is insufficient for MAPK activation in H526 cells, because overexpression of Lck or expression of the constitutively activated mutant Y505F Lck by itself did not result in significant MAPK activation in the absence of SCF (Fig. 4Citation and 5)Citation . This observation could indicate a requirement for Lck activation in proximity to the activated receptor for efficient MAPK activation, because numerous studies discussed above have demonstrated a ligand-induced association between the receptor and Src family kinases. Additional experimentation will be required to elucidate the precise mechanism by which Lck promotes MAPK activation after activation of Kit in SCLC cells.

As a by-product of the above studies we have determined that PP1 is not an absolutely specific inhibitor of Src family kinases but also efficiently inhibits Kit and potentially other class-III RTKs. During initial studies of the role of Lck in Kit signal transduction we performed an IP-kinase assay using Kit from PP1-treated cells to determine the effect of the drug on Kit activity (21) ; other investigators have also performed similar assays (43 , 44) . We chose this activity assay because it theoretically would avoid detecting potential tyrosine phosphorylation of Kit by Lck, which could complicate direct analysis of receptor tyrosine autophosphorylation. However, in the present study, when we undertook such an analysis to confirm the results of the IP-kinase assay, we found a 90–95% inhibition of Kit tyrosine phosphorylation at 5–10 µM PP1, in all likelihood too large to be accounted for by inhibition of Lck-mediated phosphorylation of the receptor. We confirmed direct inhibition of Kit activity by in vitro addition of PP1 to an IP-kinase assay. It appears that the IP-kinase assay using Kit from PP1-treated cells yielded misleading results, probably as a result of drug dissociation during the IP procedure. Thus, it is apparent that PP1 cannot be used as a probe for Src family kinase function when class-III RTK function may also be involved in the biological phenomenon being studied unless other independent confirmatory data are also obtained.

When these studies were initiated, their goal was to identify the signal transduction pathways involving Lck that are required for SCF-mediated SCLC growth (21) . Although the Ras-MAPK pathway initially seemed an excellent candidate based on its importance for the growth of other cell types and its sensitivity to Src kinase inhibition, attempts to confirm the requirement for activation of this pathway by directly inhibiting MEK failed. The lack of a requirement for MAPK activity for growth of several SCLC cell lines was initially puzzling, but a possible explanation for this result became apparent after trying to place it in the context of other genetic alterations in SCLC. The synthesis of the D-type cyclins, phosphorylation of Rb, and subsequent cell cycle progression have long been known to be responsive to growth factor stimulation (45) . However, recently several groups have linked the activity of cyclin D-dependent kinases directly to activation of the Ras-MAPK cascade in NIH 3T3 fibroblasts (46, 47, 48) . Ectopic expression of an activated form of MEK 1 could enhance cyclin D1 expression and drive the cells through S-phase if the p27Kip1 inhibitor were neutralized (47) . Conversely, DN forms of Ras and Raf could block expression of cyclin D1 and subsequent cell cycle progression in response to serum stimulation, a situation that could be rescued by ectopic expression of cyclin D1 (46) . Finally, in cells whose expression of cyclin D1 was blocked by DN Ras, structural inactivation of Rb allowed cell cycle progression (48) . This last observation was particularly revealing because it suggested that the almost universal loss of Rb function in SCLC (49 , 50) could be responsible for the observed insensitivity to PD 98059. Expression of Rb in H526 cells resulted in a partial restoration of sensitivity to the MEK inhibitor, which supports this theory. We believe that one potential reason for the incomplete restoration of sensitivity could be attributable to the effects of high levels of N-Myc expression in this cell line; overexpression of N-Myc by itself has been shown to drive cells through S phase (30) . Thus, the insensitivity to MAPK inhibition demonstrated by SCLC cells in the absence of Rb expression confirms the observations made in the 3T3 model system in a common human tumor. Although it is clear that, in an established tumor cell line, loss of functional Rb plays a dominant role in the loss of G1-S phase checkpoint regulation, it is possible that activation of the MAPK pathway could play a more important role during the pathogenesis of the tumor before the inactivation of Rb. More practically, our observations suggest that therapeutic strategies aimed at inhibition of the MAPK cascade may not be effective in tumors with a structurally inactivated Rb gene. In addition, our observations suggest the existence of one or more alternative signal transduction pathways that must be responsible for mediating SCF-stimulated growth of SCLC. Identification of these pathways, along with the precise mechanism by which Lck assists Kit in activating MAPK, will be the subject of future investigation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
The H526 SCLC cell line was grown in RPMI 1640 supplemented with 2 mM L-glutamine, with or without 10% fetal bovine serum (Life Technologies, Gaithersburg, MD); when grown in the absence of serum, 0.1% BSA (Sigma, St. Louis, MO) was added to the medium. Where indicated, serum-free medium was supplemented with recombinant SCF (Intergen, Purchase, NY) or IGF-1 (R&D, Minneapolis, MN) at the indicated concentrations. Cells were stimulated with SCF and IGF-1 after preincubation in serum-free medium overnight. The tyrosine kinase inhibitor PP1 and the MEK inhibitor PD 98059 (Calbiochem, San Diego, CA) were solubilized in DMSO; final concentration of DMSO in all of the cultures, including controls, was 0.1% in experiments using these inhibitors. Cells were pretreated for 30 min with inhibitors prior to growth factor stimulation.

Growth Assays.
Cell growth was measured using the MTT (Sigma) colorimetric dye reduction method, an assay shown to correlate very well with viable SCLC cell number under the conditions used (51) . Duplicate plates containing eight replicate wells per assay condition were seeded at a density of 1 x 104 cells in 0.1 ml of medium, and data were expressed as the change in absorbance at 540 nm over 72 h, relative to initial values obtained 3 h after plating. Tritiated thymidine incorporation in eight replicate microplate wells containing 1 x 104 cells each was determined after a 3-h incubation in medium containing 1 µCi/ml [3 H]thymidine (NEN, Boston, MA). The cells were collected on GFC filter plates using an automated cell harvester (Packard Instruments, Meriden, CT), were lysed and extensively washed with distilled water, and assayed by liquid scintillation counting.

Derivation of Cells Capable of Inducible Lck Expression.
H526 cells were transfected with the pTet-On plasmid encoding the reverse Tet repressor (Clontech, Palo Alto, CA) using electroporation parameters described previously (52) . Subclones were isolated by limiting dilution during selection with 1 mg/ml G418 (Life Technologies). The drug-resistant subclones were transiently transfected with the pTRE-Luc luciferase expression plasmid, and the IC4 subclone that had the lowest basal luciferase activity and the highest degree of doxycycline-induced activity was selected as the recipient for secondary transfections. An EcoRI fragment containing the entire murine lck cDNA (ATCC 63228) was cloned into the pTRE expression vector (Clontech) to produce the pTRE-WT Lck expression vector. The K273R and Y505F mutations were introduced into this vector using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following mutagenic oligonucleotides to produce the pTRE-DN Lck expression vector: K273R, 5'-CACGAAGGTGGCGGTGAGATCTCTGAAACAAGGGAGCATC-3' and its complement; Y505F, 5'-CACAGAGGGCCAGTTCCAGCCCCAGCCTTGATATCCCTTTCG-3' and its complement. The Y505F mutation was also singly introduced into the WT vector. In addition, to enhance translational efficiency, all of the three out-of-frame ATGs in the 5' untranslated region (53) were mutated using the same technique. All of the mutations were confirmed by sequencing. The Lck expression vectors were cotransfected with pSV2-Hygro into H526-IC4 cells, and subclones were selected by limiting dilution during culture in 600 µg/ml hygromycin (Calbiochem). The drug resistant subclones were assayed for doxycycline-inducible expression of Lck using Western blotting. The predicted kinase activities of the mutant Lck proteins were confirmed by IP kinase assays as described previously (21) . Lck expression was induced for 24 h using 2 µg/ml doxycycline (Sigma) before growth factor stimulation and MAPK assay.

IP, Immune Complex Kinase Assay, and Western Blotting.
H526 cells were lysed in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP40, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2 mM NaVO4, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 10 µg/ml leupeptin using a Dounce homogenizer with a tight-fitting pestle; protein concentrations were determined by BCA assay (Pierce, Rockford, IL). The lysate, containing 1–1.5 mg of protein, was centrifuged for 10 min at 10,000 x g to obtain a soluble postnuclear supernatant. IP was initiated by the addition of 10 µg of monoclonal anti-Kit antibody (K45; NeoMarkers, Fremont, CA), followed by incubation for 2 h at 4oC and by an additional 2 h in the presence of Protein A+G agarose. The IP was washed four times in lysis buffer and then once in PBS. The pellet was aspirated to dryness and 30 µl of kinase buffer (20 mM PIPES and 10 mM MnCl2) containing 10 µCi of [{gamma}P32]ATP was added. The kinase reaction was carried out for 10 min at room temperature and was terminated by the addition of an equal volume of 2x SDS sample-loading buffer; the reaction was then resolved on a 10% polyacrylamide gel and was analyzed by autoradiography. Western blotting was performed using standard procedures, with detection using the ECL chemiluminescent system (Amersham, Arlington Heights, IL) and visualization using a Fuji cooled CCD camera and the Aida 2.0 software package (Raytest Inc., New Castle, DE). Staining was accomplished using the following antibodies: anti-Kit, 3D6 monoclonal (Boehringer-Mannheim); antiphosphotyrosine, PY20 monoclonal (Transduction Labs, Lexington, KY); affinity-purified polyclonal antiactive MAPK (Promega, Madison, WI); polyclonal anti-ERK 1 CT (pan-ERK; UBI, Saranac Lake, NY); polyclonal anti-Lck (PharMingen, San Diego, CA); monoclonal anti-Rb (IF8; Santa Cruz Biotechnology, Santa Cruz, CA).

MAPK Assay.
The ability of immunoprecipitated MAPK to directly phosphorylate myelin basic protein was determined as described previously (54) .

Rb Phosphorylation Analysis.
H526 cells expressing doxycycline-inducible Rb protein were derived exactly as described for WT Lck-expressing cells using a full-length WT Rb cDNA cloned into the pTRE vector. Subclones capable of doxycycline-regulated expression were induced for 24 h in complete medium containing 2 µg/ml doxycycline; were placed in serum-free, phosphate-free medium containing 1 mCi/ml [32P]Pi (NEN) for 6 h; and then were stimulated with 100 ng/ml SCF after a 30-min preincubation in DMSO or 80 µM PD98059. The incubation was continued for 12 h, the cells were lysed in triple-detergent buffer (21) , and cleared lysates containing 0.5 mg of protein were used for IP using a monoclonal anti-Rb antibody (Santa Cruz). The immunoprecipitates were electrophoretically resolved and analyzed by autoradiography, phosphorimage analysis, and Western blotting. Staining with phospho-specific anti-Rb antibodies was accomplished using the PhosphoPlus antibody kit (New England Biolabs, Beverly, MA).


    Acknowledgments
 
We thank David Gewirtz (Virginia Commonwealth University, Richmond, VA) for donating the WT Rb cDNA and Sittisak Honsawek for technical assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Merit Review Award from the Department of Veterans Affairs (to G. W. K.) and by grants from the American Cancer Society (IN-105V) and the V Foundation (to P. D.). Back

2 To whom requests for reprints should be addressed, at Richmond Veterans Affairs Medical Center (111K), 1201 Broad Rock Boulevard, Richmond VA 23249. Phone: (804) 675-5446; Fax: (804) 675-5447; E-mail: GKRYSTAL{at}HSC.VCU.edu Back

3 The abbreviations used are: SCLC, small cell lung cancer; SCF, stem cell factor; DN, dominant negative; IGF-1, insulin-like growth factor-1; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; CSF, colony-stimulating factor; CSF-1R, CSF-1 receptor; IP, immunoprecipitation; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide; Rb, retinoblastoma (tumor suppressor); MAP, mitogen-activated protein; ERK, extracellular signalregulated kinase; MEK, MAP/ERK kinase; RTK, receptor tyrosine kinase; WT, wild type. Back

Received for publication 1/24/00. Revision received 5/10/00. Accepted for publication 5/10/00.


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 Materials and Methods
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