Kinases of the PAK-family have a well established role in cell polarization (Hofmann et al., 2004
). Budding yeast expresses three PAKs: Ste20, Cla4, and Skm1. To gain insight into molecular mechanisms of Ste20 action, we screened for interactors of Ste20 using the split-ubiquitin technique (Tiedje et al., 2007
). Here, we show that Sut1, a transcriptional regulator that controls sterol uptake (Bourot and Karst, 1995
; Ness et al., 2001
), forms a complex with Ste20. By using pull-down assays, we demonstrate that not only Ste20 but also Cla4 and Skm1 bind to Sut1. Sut1 localizes exclusively to the nucleus, even after strong overexpression (data not shown) (Ness et al., 2001
). Thus far, Ste20 and Cla4 have been shown to localize mainly to the plasma membrane at sites of polarized growth and to the cytoplasm, whereas the localization of Skm1 has not been reported previously (Peter et al., 1996
; Leberer et al., 1997
; Holly and Blumer 1999
). In this work, we show strong nuclear enrichment of Skm1 and we reveal an NLS. Furthermore, we identify an NLS for Ste20. The fact that a mutated Ste20, which lacks the entire NLS or a major part of it, is excluded from the nucleus under normal growth conditions, suggests that at least a small amount of Ste20 is present in the nucleus at any time. Consistently, in few cells, we observed a nuclear enrichment of wild-type Ste20. Cla4 does not contain an obvious NLS and the full-length protein is not enriched in the nucleus. Nevertheless, we observed that several Cla4 fragments that lacked the PH domain translocated into the nucleus. The PH domain is necessary for membrane association of Cla4 by binding to phosphoinositides (Wild et al., 2004
). We suggest that similar to Ste20, Cla4 that is not associated with membranes can localize to the cytoplasm and the nucleus. Therefore, it seems likely that the interaction between Sut1 and PAKs takes place in the nucleus. This notion is confirmed by our observation that the down-regulation of the Sut1 target AUS1
by Ste20 and the inhibition of PDR11
expression by Skm1 depends on the nuclear localization of these PAKs.
The expression of AUS1
, and of other genes contributing to sterol uptake is repressed under aerobic conditions. In anaerobiosis, transcriptional regulators such as Sut1 and Upc2 allow expression of these genes, which is required for sterol uptake under these conditions (Régnacq et al., 2001
; Wilcox et al., 2002
; Alimardani et al., 2004
). Here, we demonstrate that PAKs negatively regulate the expression of genes involved in sterol influx. Interestingly, we observed very different expression profiles for Ste20, Cla4, and Skm1. Ste20 regulates the expression of AUS1
, Skm1 controls AUS1
, whereas Cla4 inhibits the expression of AUS1
, and PDR11
. The reason for these different specificities is not clear.
Importantly, we demonstrate for all PAKs that the down-regulation of gene expression depends for the most part on SUT1
. Because AUS1
are target genes of Sut1 and PAKs form a complex with Sut1, it seems very likely that PAKs control gene expression via Sut1. Nevertheless, it cannot be excluded that PAKs mediate their effects on transcription through other factors such as Upc2 or the less well characterized Ecm22 (Shianna et al., 2001
) as well. PDR11
is also regulated by Cla4 and Skm1, but its transcription is controlled by Upc2 and there is no evidence that PDR11
is a target of Sut1. Further, we observed a minor reduction of AUS1
levels in the absence of SUT1
after overexpression of either STE20
. Regulation of Upc2 or other factors by Ste20 and Cla4 could account for these weak effects on AUS1
expression. We observed that sterol uptake in contrast to the regulation of AUS1
expression is independent of the Ste20 NLS. The reason for this discrepancy is not clear. However, sterol uptake is a more complex process than the transcription of an individual gene. Possibly, Ste20 contributes also to sterol uptake by other mechanisms. Ste20 could control the activity of transcriptional regulators such as Upc2 and Ecm22, which localize to the cytoplasm as well (Marie et al., 2008
Thus far, the mechanisms of sterol uptake are only poorly understood. It seems that the transcriptional regulators involved in this process act redundantly, but very little is known about Ecm22 and it is not clear how Sut1 promotes the expression of AUS1 and DAN1. Therefore, it is difficult to establish the mechanisms of inhibition of Sut1 and possibly other transcriptional regulators by PAKs. The down-regulation of AUS1 expression requires Ste20 kinase activity. Nevertheless, we did not observe Sut1 phosphorylation by PAKs using in vivo and in vitro kinase assays (data not shown). Thus, it is conceivable that PAKs control transcriptional regulators via an unknown protein.
The control of gene expression by Ste20, Cla4, and Skm1 described here is clearly distinct from the regulation of transcription by Ste20 via MAPK modules as reported previously. During mating, filamentous growth and in response to hyperosmolarity Ste20 activates the MAPK kinase kinase Ste11 by phosphorylation, which eventually results in a change of the transcriptional pattern of numerous genes (Roberts and Fink, 1994
; Wu et al., 1995
; O'Rourke and Herskowitz, 1998
; Raitt et al., 2000
). As demonstrated for AUS1
, the Sut1-mediated regulation of expression is independent of Ste11. Furthermore, it requires nuclear localization of Ste20. In contrast, the MAPK pathways promoting filamentation and the formation of a mating projection are independent of nuclear Ste20, but Ste11 is essential for these processes. Thus, the two mechanisms seem to be independent.
It has been reported that Ste20 translocates into the nucleus during hydrogen peroxide-induced cell death and directly phosphorylates histone H2B (Ahn et al., 2005
). This presumably results in chromatin condensation. However, it has not been examined whether Ste20 alters gene expression under these conditions. Whether the inhibition of a transcriptional regulator involved in sterol uptake by Ste20 also involves chromatin modification as suggested for hydrogen peroxide-induced cell death remains to be tested.
Importantly, we demonstrated that the control of genes involved in sterol import mediated by Ste20, Cla4, and Skm1 affects sterol uptake. As expected from their role as negative regulators, the deletion of either STE20, CLA4 or SKM1 results in an increased sterol influx and subsequent esterification. Furthermore, sterol uptake was markedly reduced in cells overexpressing all three PAKs.
Our data raise the question why Ste20, Cla4, and Skm1 regulate sterol uptake. The fact that all three PAKs are involved in this process, suggests that the control of sterol influx is crucial for the cell and possibly for cell polarization. Previously, we could show that Ste20 binds to Erg4, Cbr1, and Ncp1, which all catalyze important steps in sterol biosynthesis (Tiedje et al., 2007
). Interestingly, these proteins are also involved in bud site selection, apical bud growth, mating, filamentous growth, and exit from mitosis (Ni and Snyder, 2001
; Keniry et al., 2004
; Tiedje et al., 2007
). These observations highlight the importance of sterol synthesis for cell polarization. Because Ste20 plays a crucial role in all these processes as well, it seems likely that Ste20 and sterol biosynthetic proteins act in the same pathway(s). In the absence of oxygen, sterols cannot be synthesized and cells completely depend on import of sterols from the extracellular medium. Under these conditions, control of sterol uptake may be as important for cell polarization as sterol biosynthesis in aerobiosis. Therefore, it is not surprising that PAKs regulate sterol uptake. Importantly, Cla4 not only has a negative effect on sterol uptake but also down-regulates sterol biosynthesis and storage (our unpublished data). Thus, it seems that Cla4 modulates sterol homeostasis under aerobic conditions and that all PAKs are involved in sterol import under anaerobiosis. In addition, homologues of oxysterol-binding proteins, a family of proteins, which regulate synthesis and transport of sterols, were found to participate in Cdc42-dependant cell polarity (Kozminski et al., 2006
). How sterols contribute to cell polarization has been discussed controversially (Pichler and Riezman, 2004
; Alvarez et al., 2007
), and the elucidation of underlying molecular mechanisms will require further work.
In summary, we describe here a novel function for all three PAKs. They control transcription in the nucleus, which results in a change of sterol influx rate.