Haspin Modulates the G2/M Transition Delay in Response to Polarization Failures in Budding Yeast.

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Saccharomyces CDK1 phosphorylates Rad53 kinase in metaphase, influencing cellular morphogenesis.

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

Alk1 and Alk2 are two new cell cycle-regulated haspin-like proteins in budding yeast.

Haspin is a protein kinase identified in mouse and human cells, and genes coding for haspin-like proteins are present in virtually all eukaryotic genomes sequenced so far. Two haspin homologues, called Alk1 and Alk2, are present in the yeast Saccharomyces cerevisiae. Both Alk1 and Alk2 exhibit a weak auto-kinase activity in vitro, are phosphoproteins in vivo and are hyperphosphorylated in response to DNA damage. The amount and modification of the two proteins is greatly regulated during the cell cycle. In fact, Alk1 and Alk2 levels peak in mitosis and late-S/G2, respectively, and phosphorylation of both proteins is maximal in mitosis. Control of protein stability plays a major role in Alk2 regulation. The half-life of Alk2 is particularly short in G1; mutagenesis and genetic analysis indicate that its degradation is controlled by the APC pathway. Overexpression of ALK2, but not of ALK1, causes a mitotic arrest, which is correlated to the kinase activity of the protein. This finding, together with its cell cycle regulation, suggests a role for Alk2 in the control of mitosis.