CK2alpha is a novel phosphoinositide effector of PIPKIIalpha and PIPKIIbeta in DNA damage repair

Phosphoinositides (PIs) are a class of lipid second messengers that coordinate signaling events at the membrane interface. This canonical role belies the presence of rich PI metabolism within the nucleus, where emerging functions reveal pivotal contributions to oncogenesis. Despite this, effectors that transduce this lipid network are largely obscure. In particular, dysregulation of nuclear phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) and its generative kinases PIPKIIalpha and PIPKIIbeta are salient in breast cancer progression and prognosis, yet the mechanistic basis for how they drive oncogenesis remains enigmatic. Identification of the underlying pathways, therefore, remains a critical question that could inform novel therapeutic strategies. To investigate their nuclear function, we examined PI4,5P 2 dynamics by immunofluorescence (IF) and observed accumulation at gammaH2A.X-positive DNA lesions. Consistent with this, DNA damage response (DDR) assays quantifying caspase activation, growth and genomic integrity revealed that PIPKIIalpha/beta double-knockout breast cancer cells exhibited genotoxic hypersensitivity and exacerbated genomic fragmentation. These observations implicated PIPKIIalpha/beta in DDR signaling. Through mass spectrometric profiling of nuclear extracts, we identified CK2alpha as an interactor of PIPKIIalpha and PIPKIIbeta. This interaction was validated by endogenous co-immunoprecipitation and further supported by in vitro binding assays between purified recombinant proteins and proximity ligation immunofluorescence, which together revealed direct and nuclear-restricted engagement. Cells lacking PIPKIIalpha/beta selectively compromised CK2alpha activity elicited in response to DNA damage, but not mitogenic stimulation. Interrogation of CK2alpha-dependent DDR substrates further demonstrated impaired downstream signaling, substantiating PIPKIIalpha/beta control over CK2alpha in the DDR. To probe lipid signaling transduction, we performed structural, biochemical, and in silico assays that uncovered a PI-binding polybasic motif (PBM) in CK2alpha (residues 71-80) adjacent to the binding interface of its regulatory subunit CK2beta; further mutation of this PBM diminished PI4,5P 2 binding. This motivated the hypothesis that PI4,5P 2 supplants CK2beta, releasing monomeric CK2alpha, thereby redirecting its substrate specificity. Accordingly, PIPKIIalpha/beta depletion enhanced endogenous CK2alpha-CK2beta association while concurrently diminishing its interaction with the DDR-substrate XRCC1. Our findings support a model in which PIPKIIalpha/PIPKIIbeta synthesizes PI4,5P 2 following DNA damage to liberate nuclear CK2alpha from CK2beta and potentiate the DDR. Late-stage breast cancers may upregulate this pathway to mitigate otherwise lethal genetic insults and promote survival against their intrinsic genomic instability, defining a previously unrecognized nuclear PI4,5P 2 signaling axis that promotes tumor progression.