Phosphoproteomic Analysis of DNA Damage Checkpoint Kinase Signaling Reveals Unexpected Links to Actin Cytoskeletal Remodeling, Cell Migration, and Chemoresistance

The DNA damage response (DDR) is a large signaling network that senses chromatin disruption, and activates a wide range of cellular responses including transcriptional changes, cell cycle arrest, recruitment of DNA repair machinery, and induction of apoptosis or senescence if the damage is irreparable. Chemotherapeutic drugs and ionizing radiation initiate two canonical checkpoint kinase cascades mediated by ATR/Chk1 and ATM/Chk2, respectively. In addition to Chk1 and Chk2 signaling, our lab discovered that DNA damage‐induced activation of the p38/MK2 pathway was critical for sustained DNA damage checkpoint maintenance in cells lacking functional p53. MK2, the third checkpoint effector kinase required for proper DDR‐induced cell sycle arrest, shares the same substrate phosphorylation motif with Chk1 and Chk2, whose core motif is LxRxxS*/T*φ (x, amino acids; *, phosphoacceptor; φ, hydrophobic). However, though they share the same core sequence motifs for substrates, the spatial and temporal dynamics of DDR signaling indicate that each of these checkpoint kinases likely has a set of distinct substrates that contribute to the different phenotypic responses. To identify which proteins that are specifically phosphorylated by Chk1, Chk2 or MK2 in response to DNA damage, we performed large‐scale quantitative phosphoproteomic screening using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) with motif enrichment using antibodies against RxxpS/T and LxRxxpS/T, where pS/T denotes phosphoserine/threonine, corresponding to the core motif of Chk1, Chk2, and MK2 substrates. To determine which phospho‐sites are regulated differentially by each kinase following DNA damage, we used inducible knockdowns of the target kinases in U2OS human osteosarcoma cells, which were either untreated or treated with doxorubicin, a topoisomerase II poison widely used for cancer therapeutics in the clinic. Among 805 unique phosphopeptides identified, 308 phosphopeptides were up‐ or down‐regulated more than 2‐fold after doxorubicin treatment, including known DDR players such as 53BP1, EXOC4, and MCM3, as well as functionally novel proteins in terms of the DDR. By comparing the phosphopeptides identified in WT to Chk1‐, Chk2‐, or MK2‐depleted cells, 153, 98, 195 phosphopeptides were differentially phosphorylated more than 2‐fold after doxorubicin treatment, respectively, that are regarded as potential substrates of each checkpoint kinase. Surprisingly, Gene Set Enrichment Analysis revealed that the most significant enriched characteristics for phosphoproteins that are upregulated after doxorubicin in a Chk1‐ or MK2‐dependent manner is the molecular function related to actin‐cytoskeleton organization. To further investigate this, we used a high‐throughput imaging platform to systemically measure and quantify actin‐cytoskeleton rearrangements and migratory phenotypes. These experiments revealed marked changes in cell morphology and migratory behavior following low‐dose DNA damage that was blocked by chemical or genetic perturbation of either Chk1 or MK2. We show that these changes are due, in part, to alterations in focal adhesion dynamics. Finally, we show that MK2 inhibition blocks the development of a doxorubicin‐induced drug tolerant state, and reduces the emergence of a CD24 high /CD44 high chemo‐resistant population. Support or Funding Information Supported by NIH grants R01‐ES015339, R01‐GM104047, a grant from the Holloway Foundation, and a post‐doctoral fellowship from the Ludwig Cancer Research Foundation.