Centrosomes tune in to metabolic state and turn on to oxygen

Abstract

Events required for cell cycle progression, including centriole duplication and mitotic spindle formation, are obligatorily linked to the metabolic state of a cell. In this issue of show that PHD1 can act as such a sensor through proline hydroxylation of the centrosomal protein Cep192.

Cell cycle progression is driven by a timely series of events that culminates in the segregation of genetic material among daughter cells. The establishment of a functional and properly oriented bipolar spindle apparatus is pivotal to this event, and centrosomes, as microtubule organizing centers (MTOC), play a fundamental role in this process. Centrosomes are composed of two barrel-shaped centrioles and a pericentriolar matrix responsible for nucleation of microtubules. In cells that have exited the mitotic cycle, centrioles are also able to assemble cilia, antenna-like projections that sense extracellular stimuli. Defects in centrosome and ciliary structure and function are associated with human tumors and a cadre of pathologies (Hildebrandt et al., 2011). Since centrosome duplication and separation and MTOC and cilium assembly are tightly attuned to the cell cycle, it is essential that cells sense their metabolic state and translate inopportune metabolic states into growth arrest. Nevertheless, remarkably little is known about the mechanisms that link metabolic states and cell cycle progression.

It has been known for some time that reduced oxygen availability promotes transcriptional activation of a hypoxic program mediated by the hypoxia-inducible factor (HIF). Other transcription factors and a translational block are also induced through HIF-independent mechanisms, and both mechanisms combine to enforce a cell cycle block at the G1/S transition in response to hypoxia. HIF proteins are destroyed in normoxic cells through ubiquitylation by the von Hippel-Lindau (VHL) tumor suppressor, an E3 ligase, whose affinity toward HIFs is decreased by hypoxia and increased by direct proline hydroxylation (Garcia, 2009), which is implemented by a family of three prolyl-4-hydroxylases (PHD1-3). Hypoxia induces stabilization of HIF proteins through reduced activity of PHDs, since PHD activity depends upon availability of oxygen, iron, and 2-oxoglutarate (Garcia, 2009). Therefore, the abundance of PHD substrates could be directly linked to a cell's metabolic state and cellular O₂ sensing. In addition to HIF, several other proteins, including the β-adrenergic receptor (Xie et al., 2009), pyruvate kinase M2, involved in glucose metabolism (Luo et al., 2011), and human CLK-2, a DNA damage response protein (Xie et al., 2012), are known to be hydroxylated by PHD3. However, targets of PHD1 and PHD2 were unknown.

In this context, it is worthwhile to postulate that cell cycle progression and centrosome duplication could be attuned to oxygen levels within a cell, and more specifically, that hypoxia could directly put the brakes on centrosome duplication. Clearly, given their activity-dependent links to metabolic state and regulation of HIFs, PHD proteins represent strong candidates for such a sensor, and in this issue of Developmental Cell, work by Moser et al. (2013) provides support for this possibility. Interestingly, ablation of PHD1, but not PHD2 or PHD3, led to a notable delay in prometaphase of mitosis, and further analysis suggested that this delay was provoked by aberrant assembly of the mitotic spindle. Given this requirement for PHD1 in proper spindle assembly, Moser et al. searched the human proteome for mitotic spindle-associated proteins harboring a potential consensus site for PHD-mediated proline hydroxylation, and they identified the mammalian centrosomal protein, Cep192. This protein, originally identified in worms as SPD-2, resides in the pericentriolar matrix and was shown to regulate centrosome maturation and centriole duplication in mammalian cells (Zhu et al., 2008) through recruitment of proteins needed to build both centriolar and pericentriolar compartments (Carvalho-Santos et al., 2010). Since the pericentriolar material (PCM) plays a pivotal role in nucleating microtubules, Moser et al. investigated the connection of PHD1 to Cep192.

Silencing of either PHD1 or Cep192 resulted in centriolar duplication defects as well as diminished recruitment of the PCM components, γ-tubulin and pericentrin, in mitotic cells. Likewise, PHD1 ablation reduced cilia assembly, although the impact was less dramatic than Cep192 depletion. By analogy with HIFs, the authors argued that these phenotypes could be attributed to a role for PHD1 in regulating Cep192. Indeed, this was demonstrated by the fact that the two proteins stably interacted and co-localized within the pericentriolar matrix during mitosis, and PHD1 depletion led to an increase in Cep192 at centrosomes in interphase. Paradoxically, Cep192 did not accumulate in mitotic cells depleted of PHD1, an observation not fully explained here, and future experiments that examine co-localization of the proteins during the cell cycle will be required to establish a more direct relationship between these proteins. In vitro, PHD1 catalyzed hydroxylation of proline within the PHD consensus motif of Cep192. The authors developed a method to quantitatively assess the extent of this modification, and they found that ~10% of Cep192 is hydroxylated within an asynchronously growing population, although the fraction of Cep192 hydroxylated in mitosis was not investigated. Interestingly, mutation of this key proline residue (P1717A) in Cep192 led to decreased recruitment of PCM proteins in mitosis and prometaphase arrest with disorganized spindles, mimicking protein knockdown. Apart from a potential role in cell cycle regulation of Cep192, any involvement of PHD1 in regulation of this protein should be reflected by cellular oxygen availability, and this is indeed the case: hypoxic conditions led to reduced PHD activity, with concomitant increases in Cep192 and HIF levels. Surprisingly, although iron chelation led to increased Cep192 abundance at centrosomes, hypoxia led to the opposite result, suggesting additional mechanisms that restrict Cep192 localization. To complete the circuit, the authors showed that proline hydroxylation of Cep192 triggered its association with the SCF^Skp2 complex, leading to Cep192 ubiquitylation and destabilization, analogous to the relationship between HIF and VHL.

In aggregate, these findings indicate that PHD1 is required for proline hydroxylation of Cep192, which regulates the abundance and function of this protein, and this connection suggests a linkage between metabolic state, centrosome biogenesis, and cell cycle progression. Of course, every novel discovery also raises manifold questions and prompts new avenues of exploration. First and foremost, does endogenous PHD1 localize to centrosomes under normoxic and anoxic conditions, and where does PHD1 function to hydroxylate its target? It is reasonable to speculate that there are additional targets within the centrosome, cilium, and mitotic spindle that could serve as substrates for PHD proteins and additional mechanisms that respond to hypoxia by acting on protein localization as well. This speculation is supported by the fact that only a small portion of Cep192 is modified, yet ablation of PHD1 has a profound impact on centrosomes and mitosis. Intriguingly, earlier studies in Drosophila suggested that the Mps1 kinase, which plays a role in the mitotic spindle checkpoint and mammalian centrosome duplication (Fisk and Winey, 2001), was required for hypoxia-mediated metaphase arrest (Fischer et al., 2004). Thus, it is possible that the links between centrosome function and metabolic states are highly conserved. Finally, over-expression of Cep192, like depletion of Cep192 or PHD1, was detrimental, as it diminished γ-tubulin recruitment and led to abnormal centriole numbers in mitosis. These results suggest that correct levels of Cep192 are critical for normal spindle assembly and warrant a detailed investigation into expression levels and mutations in human tumors.