Abstract
Aberrant kinases contribute to cancer survival and proliferation. Here, we quantitatively characterized phosphoproteomic changes in an HBx-transgenic mouse model of hepatocellular carcinoma (HCC) using high-resolution mass spectrometry, profiled 22,539 phosphorylation sites on 5431 proteins. Using a strategy to interpret kinase- substrate relations in HCC and to uncover predominant kinases in tumors, our results, revealed elevated kinase activities of Src family kinases (SFKs), PKCs, MAPKs, and ROCK2 in HCC, representatives of which were further validated in cell models and clinical HBV-positive HCC samples. Inhibitor combinations targeting Src and PKCs or ROCK2 both synergized significantly to inhibit cell growth. In addition, we demonstrated that phosphorylation at Src Ser17 directly affects its kinase activity. Our phosphoproteome data facilitated the construction of a detailed molecular landscape in HCC and should serve as a resource for the cancer community. Our strategy is generally applicable to targeted therapeutics, also highlights potential mechanisms of kinase regulation.
Highlights
Aberrant kinase initiated dysregulation of phosphorylation signaling commonly contributes to tumor cell proliferation, survival, and migration [1, 2]
Src Ser17 is Involved in Cell Migration and Affects Rhoassociated protein kinase 2 (ROCK2) Tyr256 Phosphorylation—Our results indicated that Src is one of the predominant kinases in Hepatocellular carcinoma (HCC)
Given that Src Ser17 is involved in cell migration, we further examined ROCK2, a kinase that is involved in tumor invasion and activated by phosphorylation of the Tyr 256 residue [82, 83] and, was demonstrated to be a predominant kinase in HCC in our analysis and validation (Fig. 2C and Fig. 4B– 4F)
Summary
Aberrant kinase initiated dysregulation of phosphorylation signaling commonly contributes to tumor cell proliferation, survival, and migration [1, 2]. Kinase Substrate Relation Analysis—Overrepresented motifs were determined by analyzing the amino acid sequence motifs of regulated phosphosites in HCC with Motif-X [27] against the IPI mouse database, a minimum number of occurrences of 20 and a minimum 3.0-fold change enrichment compared with the background, the p value threshold was set to 0.000001 to maintain a low false positive rate in standard protein motif analyses.
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