Cellular that the calcium/calmodulin-dependent kinase calcineurin limits proliferation in

Cellular growth and division into
two identical daughter cells occurs through a unidirectional cell cycle. Progression
through the cell cycle is mediated by a network of transcription factors whose activity
and expression are regulated via phosphorylation by cyclin-dependent kinases
(Cdks) (Ubersax et al., 2003; Holt et all, 2009; Landry et al.; 2014). These
layers of regulation ensure that cell cycle events occur at the proper time to
prevent uncontrolled cell division and cancer. The cell cycle is also modulated
by changes in the extracellular environment to ensure that division only
proceeds when conditions are favorable for growth. Therefore, it is not
surprising that this transcriptional network is drastically rewired in response
to various environmental stresses.

For example, recent work has
shown that the calcium/calmodulin-dependent kinase calcineurin limits
proliferation in response to cellular stress (Arsenault et al., 2015).
Calcineurin does this by dephosphorylating and inactivating the S-phase
transcription factor, Hcm1 (Arsenault et al., 2015). This leads to repression
of Hcm1 target genes and cell cycle arrest (Arsenault et al., 2015).
Interestingly, calcineurin has also been shown to repress gene expression,
independent of Hcm1 target genes, at every stage of the cell cycle (unpublished
data). This suggests that calcineurin plays a more global role in stress regulation.

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Calcineurin is activated
following the influx of Ca2+ that accompanies cell wall damage,
alkaline pH, and cation imbalance (Nakamura et al., 1993; Viladevall et al., 2004;
Cyert and Philpott, 2013). It seemed plausible that the influx of calcium ions may
also activate another stress pathway. Indeed, the Hog1 mitogen-activated
protein kinase (MAPK), is activated in response to treatment with CaCl2
and maintenance of Hog1 activity is dependent on CN (unpublished data).  The HOG pathway responds to cellular
hyper-osmolarity by phosphorylating and activating the MAPK Hog1 (Saito and
Posas, 2003; Brewster et al., 1993; Maeda et al., 1994). To re-establish
osmotic homeostasis, the HOG pathway represses genes important for the G1/S
transition and induces cell cycle arrest (Bellí et al., 2001). Taken
together, these findings suggest that there may be novel cross-talk between the
two stress pathways.  

To study the possible cross talk
between the calcineurin and HOG stress pathways in S. cerevisiae we exposed synchronized cells to various osmotic
stresses (LiCl, KCl, NaCl, Sorbitol) over a fixed time course in the presence
or absence of calcineurin. These stressors have been previously shown to
perturb cellular osmolarity (Cyert and Philpott, 2013). Inhibition of
calcineurin activity was achieved using FK506 which is a known
calcineurin inhibitor (Kissinger et al., 1995). Visualization of phospho-Hog1
was achieved via Western blot. If Hog1 activity is dependent upon calcineurin,
we should expect to see rapid dephosphorylation of phospho-Hog1 in the absence
of calcineurin.

We have found that calcineurin is
required for the maintenance of Hog1 activity in response to some, but not all,
osmotic stresses. Hog1 was activated in the presence of all the stresses (CaCl2,
LiCl, KCl, NaCl, Sorbitol) we administered. However, following exposure to CaCl2
or NaCl in the absence of calcineruin, the activated form of Hog1 was quickly
degraded. This suggests calcineurin is required for maintenance of Hog1
activity in response to these stresses. Conversely, Hog1 activity was
independent of calcineurin following exposure to LiCl, KCl, and sorbitol. These
results provide the first line of evidence for cooperation between calcineurin
and HOG stress pathways. Likewise, these findings demonstrate how cell cycle
progression can be finely-tuned in response to the environment. 

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