How Growth Factors Govern Alternative Splicing

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Science Signaling  21 Aug 2012:
Vol. 5, Issue 238, pp. ec220
DOI: 10.1126/scisignal.2003517

mRNA splicing can produce proteins and regulatory RNAs with different functions, and thus regulation of splicing can alter cellular behavior. Zhou et al. elucidated a pathway that connected stimulation of cultured cells with epidermal growth factor (EGF) to the regulation of alternative splicing. The addition of EGF triggered changes in splice variants produced from a splicing reporter (E1A) transfected into human embryonic kidney 293 (HEK293) cells, which indicated that EGF altered splice site selection. Inhibition of phosphoinositide 3-kinase (PI3K) activity with Wortmannin, but not inhibition of protein kinase C (PKC), prevented the altered splice site selection in response to EGF. Cells with a constitutively active protein kinase B (Akt) or overexpressing kinases specific for the SR family of splicing factors (SRPK1 and SRPK2) exhibited the same splice site selection as EGF-treated cells, but kinase-dead mutants of Akt, SRPK1, or SRPK2 did not. Knockdown of SRPK1, SRPK2, or both with small interfering RNA (siRNA) abolished the change in splice site selection in cells treated with EGF, which indicated that both of these kinases were required for the effects of EGF on splicing. The authors used a “global splicing assay,” involving an RNA annealing, selection, and ligation assay coupled to high-throughput sequencing, to examine the effects of EGF signaling on alternative splicing of endogenous genes. Analysis of 3726 splicing events conserved between humans and mice with this global splicing assay showed the same dependency on PI3K signaling for the EGF-mediated change in splice site selection and showed very little involvement of other pathways through which EGF signals, including the Janus kinase–signal transducers and activators of transcription (JAK/STAT) pathway, the extracellular signal–regulated kinase (ERK) pathway, or the mammalian target of rapamycin (mTOR) pathway. In vitro kinase assays suggested that purified SRPK1 was a substrate of constitutively active Akt; however, a kinase-dead mutant of SRPK1 was not phosphorylated, which suggested that Akt promoted SRPK1 autophosphorylation. Mutation to alanine of residues in two putative phosphorylation sites (discovered by mass spectrometry analysis) in SRPK1 abolished the EGF-induced change in E1A splice site selection, and mutation of those residues to aspartate prevented inhibition of splicing changes by Wortmannin. EGF-treated cells had nuclear localization of SRPK1 and 2, which was blocked by Wortmannin. SRPK1 with mutations to alanine in the phosphorylation sites did not have a nuclear localization. EGF treatment of cells also led to phosphorylation of SR splicing factors, which was blocked by Wortmannin; expression of SRPK1 or 2 with mutations of their phosphorylation sites to alanine; or knockdown of SRPK1 or 2. SRPKs bind molecular chaperones the heat shock proteins Hsp70 and Hsp90 and 14-3-3 proteins. In extracts from cells at different time points after EGF stimulation, SRPK1 or 2 coimmunoprecipitated with Hsp70 at early time points, but with Hsp90 at later time points, or with an increasing amount of 14-3-3β over time; the coimmunoprecipitation with Hsp90 was inhibited by Wortmannin or overexpression of 14-3-3β. Knockdown of Hsp90 prevented EGF-induced nuclear localization of SRPK1, which suggested that Hsp90 was required for nuclear translocation. These data delineate a pathway wherein EGF stimulation activates PI3K and Akt to induce autophosphorylation of SRPKs, which changes its interaction with molecular chaperones, and enables Hsp90 to mediate SRPK translocation to the nucleus and activation of splicing factors.

Z. Zhou, J. Qiu, W. Liu, Y. Zhou, R. M. Plocinik, H. Li, Q. Hu, G. Ghosh, J. A. Adams, M. G. Rosenfeld, X.-D. Fu, The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus. Mol. Cell. 47, 422–433 (2012). [PubMed]

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