146 Global phosphoproteomic analysis of protein kinase C delta activation reveals signaling pathways involved in triggering keratinocyte growth arrest

Abstract

Activation of multiple protein kinase C (PKC) isoforms have been associated with keratinocyte (KC) cell cycle withdrawal during differentiation. To study how PKC activation may induce KC growth arrest, we evaluated the effects of the PKC agonist TPA on normal human epidermal KCs (NHEKs) and HaCaT cells, an immortalized human KC cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest, while untreated KCs underwent a 93-fold increase in cell number over 7 days (p<0.005). HaCaT cells were completely resistant to TPA-induced growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment, while HaCaT-PKCδ-EGFP cells were 80% growth inhibited by TPA over a 4 day period. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics analysis of HaCaT-EGFP and HaCaT-PKCδ-EGFP cells exposed to TPA for 30 minutes identified 667 proteins whose phosphorylation was significantly changed >2-fold, with 605 proteins having increased phosphorylation in TPA-treated PKCδ-EGFP cells. Surprisingly, only 54 of these proteins were phosphorylated at the PKCδ consensus motif, with phosphorylated motifs for the kinases MAPKAP2, AKT1 and RSK2 most abundant. Gene set enrichment analysis revealed increased phosphorylation of proteins involved in translation (eIF4, mTOR, eIF2 signaling), Rho, tight junctions and ERK/MAPK signaling. Thus, global phosphoproteomics identified PKCδ signaling pathways that can overcome the defective growth arrest in immortalized KCs and may have therapeutic benefit for the treatment of actinic keratosis and squamous cell carcinomas.