In this research project, we have revealed the following findings. When MDCK cells are cultured in matrigel, these cells form spherical cysts consisting of single-layered cells surrounding a central lumen. We found that depletion of LRRK1 by siRNA, a member of ROCO family kinase, causes the formation of multiple lumens in MDCK cells. Because proper orientation of mitotic spindle is known to be important for cystogenesis, we next examined the role of LRRK1 in this process. As a result, (1) we found that LRRK1 regulates spindle orientation downstream of PLK1, an essential mitotic kinase. PLK1 binds to LRRK1 through its polo-box domain and phosphorylates LRRK1 at Ser-1790. PLK1 phosphorylation of LRRK1 at Ser-1790 is required for CDK1-mediated activation of LRRK1 at centrosome during mitosis. (2) Activated LRRK1 then regulates spindle orientation through the formation of astral microtubules. Astral microtubules are elongated from mitotic centrosome and its formation depends on the microtubule nucleation activity of centrosome. We found that LRRK1 is important for this centrosomal microtubule nucleation activity. (3) Finally, we showed that LRRK1 phosphorylates a centrosomal protein CDK5RAP2 and promotes its association with γ-tubulin, which is an essential component for nucleating microtubules. This suggests that LRRK1 functions in the microtubule nucleation activity of centrosome via CDK5RAP2. Taken together, our findings suggest that LRRK1 plays an important role in cystogenesis through the regulation of mitotic spindle orientation downstream of PLK1 and upstream of CDK5RAP2.