Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases

Abstract

Receptor tyrosine kinases (RTKs) activate pathways mediated by serine-threonine kinases, such as the PI3K (phosphatidylinositol 3-kinase)-Akt pathway, the Ras-MAPK (mitogen-activated protein kinase)-RSK (ribosomal S6 kinase) pathway, and the mTOR (mammalian target of rapamycin)-p70 S6 pathway, that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmits signals by phosphorylating substrates on an RxRxxS/T motif (R, arginine; S, serine; T, threonine; and x, any amino acid). We developed a large-scale proteomic approach to identify more than 300 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor alpha (PDGFRalpha) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTK inhibitors (RTKIs), as well as by inhibitors of the PI3K, mTOR, and MAPK pathways, and we determined the effects of small interfering RNA directed against these substrates on cell viability. Phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at serine-305 was essential for PDGFRalpha stabilization and cell survival in PDGFRalpha-dependent cancer cells. Our approach provides a new view of RTK and Akt-RSK-S6 kinase signaling, revealing previously unidentified Akt-RSK-S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs.

Figure 1. Dual strategies to address neutral loss using monoclonal antibodies recognizing the phosphorylated RxRxxS*/T* motif

(A) Immunoaffinity purification of phosphopeptides using antibodies recognizing the RxRxxS*/T* motif with x being any amino acid. (B) Specificity of RxRxxS*/T* antibodies using SpotELISA on synthetic peptide libraries. Scale at top indicates ELISA reading; columns represent position relative to phospho-Ser/Thr at 0 position; rows represent fixed amino acid residue at the position. (C) CID spectrum of S6 peptide QIAKRRRLS*SLRASTSKSE showing unproductive neutral loss peak. (Q: Glutamine; I: Isoleucine; A: Alanine; K: Lysine; R: Arginine; L: Leucine; S: Serine, S*: phospho-Serine; T: Threonine; E: Glutamate. (D) Dual strategy to address neutral loss. (E) ETD spectrum of the S6 peptide shown in panel C. (F) CID spectrum of truncated S6 peptide after tryptic digest LS*SLRASTSKSE following IAP. The inset of 10x enlarged image shows efficient backbone fragmentation of the peptide.

Figure 2. Effects of RTKIs and pathway inhibitors on Akt-RSK-S6 kinase signaling

(A) Strategy of probing signaling pathways with different inhibitors. Wort, wortmannin; Rapa, rapamycin. (B) Immunoblotting of three cancer cell lines treated with the indicated inhibitors. (C) Immunoblotting showing the effect of inhibitors on Akt, RSK, S6, and ERK phosphorylation. (D) Confirmation of MS/MS results by immunoblotting wih phosphospecific antibodies. Actin was used as loading control.

Figure 3. Comparison of sites and proteins identified using RxRxxS*/T* and pY antibodies

(A) Amino acid distributions in singly phosphorylated phosphoserine peptides identified using the indicated RxRxxS*/T* antibody compared to known Akt substrates from the literature. Phosphopeptide sequences containing the full motif RxRxxS*/T* were aligned with phospho-serine/threonine at the 0 position, and the frequency of each amino acid at each flanking position was calculated and plotted. (B) Protein containing RxRxxS*/T* sites identified with 110B7 antibody arranged by protein type. Only the top 11 classes were shown, the rest were classified as other. (C) Proteins containing pY sites identified using the phosphotyrosine (pY-100) antibody arranged by protein type.

Figure 4. Pathway mapping downstream of EGFR, c-Met, and PDGFRα using RTKIs and pathway inhibitors

(A) Overlap of RxRxxS*/T* protein phosphorylation inhibited by Gleevec, gefitinib, and SU11274 in H1703, H3255, MKN45 cells, respectively. (B) Proteins whose phosphorylation was decreased by corresponding RTK inhibitors in all three cancer cell lines, phosphorylation sites and fold change in phosphorylation status relative to untreated cells are shown. (C) Venn diagrams of protein phosphorylation decreased by inhibitors in three cancer cell lines. (D) Enrichment of GO terms among the targets of each inhibitor (relative to the total numbers in their respective categories) was determined using the Pathway Studio program by Fisher’s Exact Test. Significance is represented as the −log(P value); the significance threshold is 1.301 = −log (P = 0.05).

Figure 5. Connections iidentified using RTKIs and pathway specific inhibitors

Proteins inhibited by two or more RTKIs and the indicated pathway inhibitor(s) are grouped by protein function. Color coding indicates sensitivity to pathway inhibitor.

Figure 6. Proteins subject to both RxRxxS*/T* and tyrosine phosphorylation

(A) Overlap of proteins showing greater than 2.4 fold change on RxRxxS/T and tyrosine phosphorylation sites. (B) Functional categorization of proteins phosphorylated on both RxRxxS/T and tyrosine sites downstream of RTKs. (C) Examples of protein types phosphorylated on both RxRxxS/T and tyrosine sites downstream of RTKs.. (D) Tyrosine and RxRxxS/T phosphorylation sites on individual proteins phosphorylated downstream of RTKs are often clustered in close proximity as shown for LOM7 at Ser⁸⁰⁵ and Tyr⁸²⁶, Ser^1493/1501 and Tyr^1401/1430.

Figure 7. Functional assay of AGC-family substrates

(A) Cell viability after siRNA transfection. H1703 cells were collected 60 hrs after transfection with the indicated siRNA, stained with Trypan Blue and counted. Numbers are mean ± SD from three different wells. Controls were transfected with scrambled siRNA. Results are representative of three independent experiments. (B) Immunoblotting of H1703 cells transfected with siRNAs. 60 hrs after transfection, cell lysates were made and probed with PARP antibody. (C) Immunoblotting of H1703 cells transfected with siRNAs. Cell lysates were made as in 7b and probed with PDGFRα antibody. (D) Immunoblotting of H1703 cells transfected with PDGFRα and SGTA siRNAs. 60 hrs after transfection, cell lysates were collected and probed with the antibodies indicated. (E) Immunoblotting of H1703 transfected with siRNAs. Cell lysates were made as in 7b and probed with the antibodies indicated. (F) Cell viability of H1703 cells transfected with siRNAs. siRNA transfected cells were collected and counted as in 7a. (G) Half life of PDGFRα in H1703 cells transfected with SGTA siRNA or treated with Gleevec. 48 hrs after siRNA transfection or overnight treatment with Gleevec (1 mM), cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 0.1 mg/ml) for the indicated time and cell lysates were collected and probed with PDGFRα antibody. (H) SGTA S305 phosphorylation is required for SGTA/PDGFRα interaction. Cells were transfected with Flag-tagged wildtype SGTA (WT) or S305A mutant (SA). 32 hrs after transfection, cells were treated with Gleevec (1 uM) for 2.5 hrs and cell lysates were immunoprecipitated with Flag (left panel) or PDGFRα (right panel) and probed with antibodies indicated. (I) SGTA Ser³⁰⁵ is required for PDGFRα stability. SGTA siRNA knockdown cells were complemented with murine SGTA WT, or the S307A mutant. Cell lysates were collected and probed with antibodies indicated. (J). Gleevec sensitivity of H1703 cells transfected with SGTA siRNA. 32 hrs after SGTA siRNA transfection, cell were treated with Gleevec (1 uM) for 24 hrs and cells were counted as in 7a. Results are representative of 2 independent experiments. (K). A model for the effect of putative Akt substrates on PDGFRα stability, cancer cell growth and death.