The Snf1 kinase is activated by an upstream kinase (21
). However, genetic screens have failed to identify mutations in any protein kinase gene other than SNF1
that produces a Snf−
phenotype. We have argued previously that the most likely explanation for these findings is that more than one kinase is capable of activating Snf1 in vivo. In this report, we present the following evidence that the Pak1 kinase is one of the Snf1-activating kinases. First, Pak1 associates with the Snf1 kinase complex in vivo and the association is greatly enhanced under growth conditions that promote Snf1 activation (Fig. .). This result puts Pak1 in the right place at the right time. Second, Pak1 kinase activates Snf1 kinase in vitro (Fig. ). Third, purified Pak1 is able to phosphorylate Snf1 protein on threonine 210 within the activation loop (Fig. ) and an increased PAK1
gene dosage increases the level of threonine 210 phosphorylation in vivo. Finally, Snf1 kinase purified from pak1
Δ mutant strains has a lower specific activity than Snf1 kinase purified from PAK1
mutant strains (Fig. ). Taken together, these data demonstrate that Pak1 is a Snf1-activating kinase. Our current model for the phosphorylation events occurring in the Snf1 kinase complex is presented in Fig. .
FIG. 8. Model of Pak1-dependent activation of Snf1 kinase. In the presence of a high glucose concentration, Snf1 kinase is in a complex with the Reg1 protein, which recruits protein phosphatase Glc7 (not shown). The Reg1/Glc7 complex dephosphorylates Snf1 threonine (more ...)
The one troubling aspect of this work is that the pak1
Δ allele does not produce any detectable Snf phenotype. We cannot formally rule out the possibility that Pak1 is a kinase whose in vivo function is to activate kinases other than Snf1. However, the findings that Pak1 associates with Snf1 in vivo under conditions in which Snf1 is active and that Pak1 promotes phosphorylation of Snf1 threonine 210 in vivo argue against this interpretation. Furthermore, we show that deletion of PAK1
suppresses the phenotypes associated with reg1
Δ. The Reg1-Glc7 PP1 phosphatase is known to act in opposition to Snf1 kinase. Deletion of REG1
results in constitutively activated Snf1. The observation that the pak1
Δ mutation suppresses the phenotypes caused by the reg1
Δ mutation argues strongly that Pak1 is one of the Snf1-activating kinases in vivo. We favor the interpretation that activation of Snf1 kinase is a redundant function that is shared between Pak1 kinase and one or more other kinases. Hartwell and colleagues have pointed out that redundancy is an important source of buffering genetic variation (10
). Many yeast genes have one or more paralogues that may mask phenotypes caused by mutations. In the case of Pak1, the closest relative is Tos3 (16
); however, the pak1
Δ mutant strain is also Snf+
(data not shown). Identification of the other putative Snf1-activating kinase(s) would settle the question unambiguously and is a high priority for our future studies. An increased PAK1
gene dosage leads to increased phosphorylation of Snf1 threonine 210 under glucose-repressing conditions but does not lead to activation of Snf1 kinase. Previously, we showed that activation of Snf1 kinase is a two-step process (21
). We conclude that phosphorylation of Snf1 threonine 210 is required but not sufficient for Snf1 activation.
gene was first isolated in a screen for high-copy suppressors of the temperature sensitivity phenotype caused by the cdc17-1
(also known as CDC17
) encodes one subunit of the alpha DNA polymerase-primase complex. An increased PAK1
copy number partially suppresses the temperature sensitivity phenotype caused by several alleles of POL1
. However, a direct connection between Pak1 and DNA polymerase alpha was not established. Furthermore, an increased PAK1
gene dosage did not suppress mutations in other DNA polymerases or even other mutations affecting DNA polymerase alpha. The ability of PAK1
to suppress the cdc17-1
allele required a functional kinase domain. We show here that Pak1 kinase is able to phosphorylate the activation loop threonine of Snf1 kinase (Fig. ). In addition, the mammalian homologues of Pak1 also activate downstream kinases by phosphorylation of activation loop residues. These observations suggest that Pak1 suppressed the cdc17-1
allele by activating a downstream kinase. It is unlikely that Pak1 is devoted to Snf1 activation. Some other in vivo target of Pak1 might provide an explanation of its ability to suppress mutations affecting the alpha DNA polymerase-primase complex.
Independent experiments demonstrate that Pak1 associates with the Snf1 kinase complex in vivo. In mass spectrometry studies of proteins associated with TAP-tagged Snf1 and TAP-tagged Snf4, both detected Pak1 as a complex member (6
). We used myc-tagged Pak1 and HA-tagged Snf1 to demonstrate the association of these proteins by coimmunoprecipitation (Fig. ). In the course of that experiment, we found that Pak1 accumulation increased under conditions of glucose limitation. A similar increase in Pak1 mRNA was not detected in microarray experiments (3
), suggesting that the increase in Pak1 protein abundance may reflect posttranscriptional regulation. Also, a shift in the SDS-polyacrylamide gel mobility of the Pak1 protein in response to glucose limitation was observed, suggesting that the activity or abundance of Pak1 may be regulated by a posttranslational modification.
The mammalian homologue of Snf1 kinase, AMPK, is the subject of intense study, in part because of its potential as a target for treatment of type II diabetes (12
). Biochemical studies have shown that AMPK is activated by a distinct protein kinase designated AMPKK (11
). The identity of AMPKK has not been determined. Our finding that Pak1 kinase is one of the Snf1-activating kinases in yeast suggests that the mammalian homologue of Pak1, CaMKK-β, may play a similar role in mammalian cells. Biochemical properties of AMPKK and CaMKK-β were used to argue that these enzymes are distinct (13
). However, this study based its conclusion on efficiency of activation and relied on partially purified fractions containing AMPKK and CaMKK-β. In some studies, CaMKK-β has been used as a surrogate AMPKK since recombinant CaMKK-β is able to phosphorylate the activation loop threonine of AMPK in vitro (8
). Full activation of AMPK may involve more than one phosphorylation event (31
). Perhaps efficient activation of AMPK requires the action of more than one kinase enzyme, a possibility that may explain the difficulty with the purification and identification of a single AMPKK. Our data suggest that CaMKK-β may participate in the activation of AMPK in vivo by phosphorylation of the activation loop threonine.