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the effects of a panel of CaM inhibitors on EGFinduced proton efflux in podocytes. The results in Figure 4A demonstrate that W 7, fluphenazine, Decitabine and ophiobolin A, each inhibited EGF induced increases in ECAR by 60 . Because none of those agents decreased the basal levels of proton efflux in podocytes, the results are most consistent with EGF activation of NHE 1. Because previous studies from our laboratory demonstrated that Jak2 is vital for NHE 1 activation by hypertonicity and by Gq coupled receptors , we analyzed the effects of a Jak2 inhibitor, AG490, on EGF induced activation of NHE 1 in podocytes. AG490 inhibited EGF induced increases in ECAR by 50 . The EGFR tyrosine kinase inhibitor AG1478 also inhibited ECAR in podocytes that were stimulated with EGF by 95 .
These outcomes assistance the involvement of Jak2 as well as the EGFR in the EGF induced increases in ECAR. EGF increases formation of complexes of Jak2 and NHE 1 with CaM To further examine a function for Decitabine Jak2 in EGF induced signaling, we determined whether or not EGF stimulates the formation of signaling complexes in between Jak2, NHE 1, and CaM. To explore this possibility, we performed co immunoprecipitation experiments working with cell lysates from podocytes pretreated with car or with inhibitors of Jak2 or EGFR tyrosine kinases. Figure 5A shows that CaM was present in Jak2 immunoprecipitates, and that the amount of CaM present in these immunoprecipitates was doubled soon after EGF stimulation. Pretreatment of cells having a Jak2 inhibitor, AG 490 considerably decreased the amount of CaM in Jak2 immunoprecipitates, whereas pretreatment with an EGFR kinase inhibitor, AG1478 did not have such effect.
This result suggests that EGF induced Jak2 activity is necessary for formation from the complex in between Jak2 and CaM. Additionally, Figure Doxorubicin 5B shows that there was a marked boost in the amount of CaM in NHE 1 immunoprecipitates soon after therapy with EGF. In contrast, there was not an improved formation of complexes in between Jak2 and NHE 1 in podocytes soon after therapy with EGF . Pretreatment of cells having a Jak2 inhibitor, AG490 or EGFR kinase inhibitor, AG1478 decreased the amount of CaM in NHE 1 immunoprecipitates. The latter result suggests that both EGFR kinase activity and Jak2 activity are required to induce formation of a complex in between CaM and NHE 1.
EGF Induces Tyrosine Phosphorylation of Jak and CaM To be able to examine further the signaling mechanisms involved in the activation of NHE 1 by EGF, we next considered that EGF could stimulate tyrosine phosphorylation of CaM. The data presented in Figure 6 demonstrate that EGF PARP improved the amount Doxorubicin of EGFR in phosphotyrosine immunoprecipitates, and that this effect is unchanged in the presence of Jak2 inhibitor, but is fully abolished soon after pretreatment with AG1478. This result demonstrates that AG1478 effectively inhibits intrinsic EGFR tyrosine kinase activity in podocytes. Figure 6 shows that EGF induces tyrosine phosphorylation of Jak2, that is inhibited by pretreatment with AG 490, but not with AG 1478. These outcomes provide robust evidence that EGF induces tyrosine phosphorylation of EGFR and Jak2 through auto phosphorylation of these kinases, and also demonstrate that AG 490 and AG 1478 were efficient under our experimental conditions.
The results also suggest that EGFR kinase activity is just not required for Jak2 Decitabine activation by EGF. Figure 6 demonstrates that EGF increases the amount of CaM in phosphotyrosine immunoprecipitates and that this effect could be considerably decreased by pretreatment of cells with AG 490, but not with AG 1478, suggesting that tyrosine phosphorylation of CaM is induced by Jak2, and doesn't demand EGFR kinase activity. In that regard, we demonstrated previously that CaM is often a bona fide substrate for Jak2 . DISCUSSION What is new about this work is that we have demonstrated that EGF activates NHE 1 via the intermediary actions of Jak2 and CaM in renal podocytes.
The work expands recent studies demonstrating that hypertonicity and Gq coupled receptors Doxorubicin activate NHE 1 in various cell sorts via a pathway involving sequential phosphorylation and activation of Jak2, tyrosine phosphorylation of CaM, CaM binding to NHE 1, and activation of NHE 1. The current work is significant in that we have demonstrated that a prototypical receptor tyrosine kinase utilizes this pathway along with a second pathway, both of which are required for full activation of NHE 1; refined the previously identified pathway as follows: EGF EGFR Jak2 activation tyrosine phosphorylation of CaM CaM binding to NHE 1 activation of NHE 1; characterized a second activation pathway as follows: EGF EGFR EGFR kinase activation association of CaM to NHE 1 activation of NHE 1 . We also have identified mRNAs for several isotypes of plasma membrane NHEs, and for EGFR related subunits, in renal podocytes. Because podocytes happen to be implicated as playing important roles in the initial stages of several glomerular diseases, this new data may well h