Systems-wide analysis of K-Ras, Cdc42, and PAK4 signaling by quantitative phosphoproteomics

Abstract

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.

Fig. 1.

Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.

Fig. 2.

Depth of the detected phosphoproteomes. The number of identifications was similar between different phospho-motif antibody enrichment methods ranging from 617 to 827 phosphopeptides after filtering out duplicates. Around 67% of the mapped phosphosites were novel in reference to the PhosphoSitePlus database. Overall, 68% of the identified peptides were singly phosphorylated, whereas 32% of the peptides carried multiple phosphorylation sites.

Fig. 3.

Quantitative phosphoproteomic analysis of K-Ras, Cdc42, and PAK4 perturbation. A, The numbers of phosphorylated peptides (-proteins) with threefold intensity changes induced by K-Ras activation, Cdc42 activation, or PAK4 knockout were in a comparable range. The number of regulated phosphoproteins is given in parentheses. Up-regulated phosphopeptides are boxed in red, whereas down-regulated phosphopeptides are boxed in green. Density plots illustrate the distributions of logarithmized phosphopeptide intensity ratios for given comparisons. Venn diagrams depict the overlap in regulated phosphopeptides (upper diagram) and phosphoproteins (lower diagram). WT: wild-type; KO: knockout; pLPC: empty vector control. B, Clustering (using Ward's method) of the 100 most variable phosphorylated peptides based on measured intensities elucidated different regulation patterns. C, Biological processes that were significantly overrepresented in the set of regulated proteins based on Gene Ontology annotation. The expected number of proteins associated with a certain cellular process was derived from the total set of identified phosphoproteins. Enrichment of GO terms among the targets of K-Ras, Cdc42 and PAK4 was determined using the R package GOstats.

Fig. 4.

Validation of PAK4-mediated phosphorylation events. Lysates derived from NIH3T3 wild-type (WT) or PAK4 knockout (KO) cells expressing K-Ras V12, Cdc42 V12, or a pLPC empty vector control were subjected to immunoblot analysis.

Fig. 5.

Upstream regulation of PAK4-mediated phosphorylation events. A, The regulation of select phosphoproteins by K-Ras activation, Cdc42 activation, or PAK4 knockout is depicted. “Regulation observed in distinct phosphopeptides” indicates that the abundances of multiple distinct phosphopeptides of a particular phosphoprotein are modulated by pathway perturbation. “Diverse regulation” indicates that the abundances of distinct phosphopeptides are differentially regulated by pathway perturbation. “Reverse regulation” depicts phosphoproteins for which phosphopeptide intensity changes induced by PAK4 knockout are reversed by K-Ras V12 or Cdc42 V12 expression. B, Phosphorylated peptides with minimum threefold intensity changes induced by PAK4 knockout in pLPC control samples were defined as ‘reversed’ by K-Ras V12 or Cdc42 V12 expression, if there was no intensity change (less than threefold) or opposite changes between K-Ras/Cdc42 activated PAK4 knockout and pLPC treated wild-type samples. In addition the intensity ratio measured in K-Ras/Cdc42 V12 versus pLPC control samples (both with PAK4 knockout) was required to be converse (minimum twofold).