c Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with ␤1 integrin. This interaction between myosin X-FERM and ␤1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-␤1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation. Spindle orientation defines the axis of cell division and is important for asymmetric cell division, tissue morphogenesis, and organogenesis. Hertwig first identified cell shape as one of the determinants of spindle orientation (1). According to the Hertwig rule, cells divide along their long axis, which is considered the default mechanism for spindle orientation (2-4). Cells proliferating in culture dishes or in tissues in vivo, however, preferentially divide according to internal or external polarity cues that include asymmetrically localized polarity proteins as well as cell-cell and cell-extracellular matrix (ECM) adhesion (reviewed in references 4-8). Although in-depth studies have identified the mechanisms regulating spindle orientation within the context of cell-cell adhesion and the axis of cell polarity, the mechanisms underlying cell-ECM adhesion-dependent spindle orientation remain poorly characterized.We have previously shown in nonpolarized adherent cells, such as HeLa cells, that integrin-mediated cell-ECM adhesion aligns the mitotic spindle along the plane of the ECM, ensuring both daughter cells remain attached to the ECM following cell division (9). This mechanism requires the actin cytoskeleton, astral microtubules, the microtubule plus-end-tracking protein EB1, the actin motor protein myosin X, and the plasma membrane phospholipid phosphatidylinositol 3,4,5-triphosphate (9-11). Following a genome-wide RNA-interference (RNAi) screen of human kinases, we identified ABL1 tyrosine kinase as a novel regulator of spindle orientation (12). We also identified PCTAIRE-1 kinase (PCTK1; also referred to as Cdk16) as a strong candidate regulator of spindle orientation.PCTK1 to PCTK3 are highly conserved serine/threonine kinases that belong to the cyclin-dependent kinase (CDK) family of protein kinases (13,14). These kinase...