CAC3 (MSI1) Suppression ofRAS2^G19V Is Independent of Chromatin Assembly Factor I and Mediated by NPR1

Abstract

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability

“…One interpretation of these results is that the increased GCR rate of the cac1 single mutant and the cac1 cac3 double mutant may in part be due to the aberrant activity of subcomplexes of CAF-I containing CAC2 and possibly ASF1 (see below). It should be noted that other studies have also suggested that the different CAF-I subunits may also have distinct functions in addition to a shared function implied by their presence in the CAF-I complex (43)(44)(45)(46)(47), and defects in these different functions could also contribute to the differences between the effects of the cac1, cac2, and cac3 mutations seen here.…”

“…Interestingly, induction of a dominant-negative CAC1 in mammalian cells induced phosphorylated histone H2Ax foci (31). It should also be noted that other studies have suggested that CAC1-CAC3 all seem to have distinct functions in addition to a common function (43)(44)(45)(46)(47), and it is known that a portion of CAC3 does not copurify with the CAF-I complex (49,70). Thus, it is also possible that these differences in function could contribute to the differences in the effects of the cac1, cac2, and cac3 mutations seen here.…”

“…Recently it was shown that expression of a dominant-negative CAC1 protein in mammalian cells seems to induce DNA damage during S phase and activation of S-phase checkpoints (31). Interestingly, several genetic studies have suggested that CAC1-CAC3 may also have independent functions (43)(44)(45)(46)(47). RCAF consists of antisilencing function 1 (ASF1) and histones H3 and H4 and can also promote assembly of nucleosomes in vitro (36).…”

Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

“…Although the precise regulatory mechanism via which Msi1 functions in the Ras/ cAMP-signaling pathway in budding yeast is not completely understood, several lines of evidence indicate that Msi1 operates either between the Cyr1 adenylyl cyclase and protein kinase A (PKA) or downstream of PKA. MSI1 overexpression suppresses the heat shock sensitivity caused by the deletion of two phosphodiesterases, Pde1 and Pde2, or by an activated TPK2 allele but not by the deletion of the Bcy1 regulatory subunit of PKA, suggesting that Msi1 downregulates Ras/cAMP signaling by inhibiting PKA in a Bcy1-dependent manner (21,46). Supporting this, Msi1 does not inhibit intracellular cAMP synthesis (46).…”

“…Interestingly, however, Msi1 also does not affect total PKA catalytic activity, indicating that Msi1 may affect the subcellular localization of PKA (46). In addition, Msi1 was found to negatively regulate the Ras/cAMP pathway by sequestering and inactivating the Npr1 serine/threonine kinase, which is known to promote or antagonize the ubiquitin-mediated degradation of several nutrient transporters (21). Indeed, MSI1 overexpression phenotypes are strikingly similar to those of an npr1⌬ mutant.…”