LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2

Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucine-rich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen.¹ PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor.² However, the role of LRRK1 in mitosis has remained unknown.

Recently, we characterized the roles of PLK1 and LRRK1 in spindle orientation.³ We found that PLK1 interacts with LRRK1 through its Polo-box domain and phosphorylates LRRK1 at Ser-1790, an event that is required for cyclin-dependent kinase 1 (CDK1)-mediated activation of LRRK1 at centrosomes. CDK1 phosphorylates LRRK1 at Thr-1400, which is located in the activation loop of LRRK1, turning on LRRK1 kinase activity. Thus, LRRK1 is activated by the sequential phosphorylation of 2 mitotic kinases, PLK1 and CDK1. Importantly, this PLK1-dependent activation of LRRK1 is required for the regulation of mitotic spindle orientation. We showed that LRRK1 regulates spindle orientation in a manner dependent on its kinase activity. Furthermore, the spindle misorientation caused by PLK1 inactivation is rescued by expression of the phospho-mimicking mutant LRRK1(S1790D), but not the non-phosphorylatable mutant LRRK1(S1790A), suggesting that PLK1 phosphorylation of LRRK1 Ser-1790 regulates the orientation of the mitotic spindle. Thus, LRRK1 regulates spindle orientation downstream of PLK1.

Correct mitotic spindle orientation depends on the interaction of astral microtubules (MTs) with the cell cortex and MT nucleation activity of the mitotic centrosome is indispensable for astral MT formation. γ-tubulin ring complex (γTuRC), which is a large multi-protein complex containing γ-tubulin, is known to be essential for MT nucleation. In principle, MT nucleation of centrosomes could be regulated at one or more steps including the assembly of γTuRC, its localization at centrosomes, and its subsequent nucleating activity.⁴ Since LRRK1 depletion causes a reduction in MT nucleation of mitotic centrosomes, we examined the role of LRRK1 in each of these steps. As one binding partner of LRRK1, we identified the centrosomal protein CDK5RAP2, a human homolog of Drosophila Centrosomin (Cnn). CDK5RAP2 is required for centrosome MT nucleation activity and shares homology with Drosophila Cnn in 2 of its domains, CM1 and CM2: CM1 is involved in the association with γ-tubulin, while CM2 is important for centrosomal localization.⁵ Interestingly, LRRK1 associates with the N-terminal region of CDK5RAP2, which includes the CM1 domain, and phosphorylates CDK5RAP2 at Ser-140, which is located close to the CM1 domain. Since the CM1 domain of CDK5RAP2 directly binds to γTuRC, it has been thought that CDK5RAP2 may facilitate the recruitment of γTuRC to mitotic centrosomes. However, although knockdown of LRRK1 results in a reduced interaction between the CM1 domain of CDK5RAP2 and γ-tubulin, we found that it does not affect γ-tubulin recruitment to mitotic centrosomes per se.

CDK5RAP2 has also been proposed to function in the activation of γTuRC. In animal cells, the majority (80%) of γTuRCs proteins in the cytoplasm are inactive, and the mechanism by which their MT nucleating function is activated at the centrosomes during mitosis is largely unknown. The MT nucleating activity of γTuRC is enhanced when bound by the CM1 domain of CDK5RAP2. It has been postulated that this activation of γTuRC nucleation activity occurs via a conformational change induced by the binding of CDK5RAP2.⁶ Interestingly, we found that LRRK1 is required for CM1-induced ectopic MT nucleation via phosphorylation of Ser-140. Mechanistically, we have demonstrated that LRRK1-dependent phosphorylation of CDK5RAP2 Ser-140 promotes the formation of a CDK5RAP2–γTuRC complex. This step appears to be critical for stimulating the MT-nucleating activity of γTuRC.

In the current study, we have established that the PLK1/CDK1–LRRK1–CDK5RAP2 signaling pathway is a regulator of centrosome MT nucleation and spindle orientation during mitosis.³ In humans, CDK5RAP2 is also known as an autosomal recessive primary microcephaly gene, mutation of which causes small brain size resulting from deficient neuron production in the developing cerebral cortex.⁵ Importantly, it was recently reported that a mouse harboring an in-frame deletion within the CM1 motif of CDK5RAP2 suffers from microcephaly.⁷ Our work should therefore provide new mechanistic insights into the regulation of CDK5RAP2 by LRRK1 through the CM1 motif (Fig. 1).

Figure 1.

Proposed model in which LRRK1 is activated by sequential phosphorylation of PLK1 and CDK1 at the centrosome and then phosphorylates CDK5RAP2, leading to the promotion of CDK5RAP2–γTuRC complex formation and the activation of γTuRC. Consequently, spindle orientation is properly regulated by the interaction between the cell cortex and astral MTs emanating from the centrosome at metaphase.