Asc1p/RACK1 is a major player in cellular signaling

Asc1p/RACK1 interacts with numerous players of signaling pathways in various eukaryotic organisms. As a ribosomal protein Asc1p/RACK1 was attributed a function as a molecular link between cellular signaling and translation. However, it not only links signals to the translational machinery, but also mediates the cross-talk between different signaling pathways, which is outlined in the following sections.

1.5.2.1 Asc1p/RACK1 interacts with components of the cAMP/PKA signaling pathway Asc1p is a Gβ-like protein, but is missing the N-terminal coiled-coil domain that is required for Gγ-binding and thus characteristic for canonical Gβ-subunits. Still, in S. cerevisiae Asc1p was described to function as Gβ-subunit in the cAMP/PKA pathway for glucose signaling (Zeller et al., 2007). It physically interacts with the glucose receptor-associated Gα-protein Gpa2 (Fig. 5) specifically in its GDP-bound form and inhibits the guanine nucleotide exchange (Zeller et al., 2007). The G-protein controls the activity of the adenylate cyclase Cyr1p which produces cAMP upon pathway activation. cAMP subsequently activates the protein kinase A (PKA) resulting in the activation of the transcription factor Flo8p and others required for invasive and pseudohyphal growth (Fig. 5). The physical interaction between Asc1p and Cyr1p causes decreased cAMP production upon glucose stimulation (Fig. 5; Zeller et al., 2007). In contrast, the Asc1p orthologue Gib2p in C. neoformans positively regulates cAMP levels. Like Asc1p, Gib2p functions as the Gβ-subunit in cAMP signaling (Palmer et al., 2006). It physically interacts with the Gα-subunit Gpa1p and additionally with the Gγ proteins Gpg1p and Gpg2p.

Furthermore, a direct interaction with the downstream signaling target Smg1p was observed (Palmer et al., 2006).

Further components of the cAMP/PKA signaling pathway are phosphodiesterases required to lower cAMP levels by breaking phosphodiester bonds within the second messenger. Human RACK1 specifically interacts with the PDE4 isoform PDE4D5 (Steele et al., 2001; Yarwood et al., 1999). This interaction enhances the binding affinity of PDE4D5 to cAMP in membrane fractions of HEK293 cells (Bird et al., 2010). Via binding to RACK1 PKCα can phosphorylate and activate PDE4D5 (Bird et al., 2010) demonstrating that RACK1 mediates a cross-talk between cAMP and PKC signaling.

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Fig. 5: Asc1p interacts with components of the S. cerevisiae cAMP/PKA pathway and MAPK cascades. The Asc1 protein physically interacts with the Gα-subunit Gpa2p and with the adenylate cyclase Cyr1p of the glucose response pathway. Thereby, it negatively affects cAMP signaling. Asc1p further binds the MAP4K Ste20p of the MAPK pathway responding to starvation and influences Kss1p (MAPK) phosphorylation. Both pathways regulate pseudohyphal and invasive cell growth. The MAP4K Ste20p is also part of the mating and osmotic stress response pathway. Asc1p-depletion results in a decreased phosphorylation level of the osmotic stress MAPK Hog1p. Within the cell wall integrity pathway an interaction of Asc1p with the MAPK Slt2p was described and Slt2p-phosphorylation is increased in Asc1p-depleted cells. Pathway information were obtained from Breitkreutz et al., 2010, Schmitt et al., 2017, Zeller et al., 2007, and the KEGG database (http://www.genome.jp/kegg).

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1.5.2.2 Asc1p/RACK1 influences MAPK pathways by differential protein-protein interactions

Asc1p/RACK1 is implicated in several MAPK cascades and physically interacts with components of these pathways in different organisms. The signal transduction pathway regulating cell wall integrity in S. cerevisiae comprises the yeast protein kinase C (Pkc1p) and a downstream MAPK module including the MAPK Slt2p (Fig. 5). Asc1p physically interacts with the MAPK and its absence results in Slt2p hyperphosphorylation (Fig. 5; Breitkreutz et al., 2010; Chasse et al., 2006). Accordingly, cell wall integrity is disturbed in cells lacking Asc1p (Rachfall et al., 2013; Valerius et al., 2007). Furthermore, Asc1p binds Ste20p which locates upstream of the MAPKs Fus3p, Kss1p and Hog1p controlling the pheromone response pathway, invasive/pseudohyphal growth and the osmotic stress response, respectively (Fig. 5;

Zeller et al., 2007). Asc1p depletion causes an increase in Kss1p phosphorylation and a significant reduction in Hog1p phosphorylation at sites required for its activity (Fig. 5; Schmitt et al., 2017; Zeller et al., 2007). Accordingly, Asc1p-depletion results in diminished adhesion and pseudohyphae formation and increased sensitivity against osmotic stress (Melamed et al., 2010; Valerius et al., 2007).

In A. thaliana, RACK1 functions as scaffold protein in a similar manner as the yeast Ste5 protein. RACK1 scaffolds the MAPK module that answers to pathogen-secreted proteases. It interacts with the MAP3K MEKK1, the redundant MAP2Ks MKK4 and MKK5, and the MAPKs MPK3 and MPK6 and facilitates their communication (Cheng et al., 2015).

Furthermore, RACK1 interacts with AGB1, the Gβ-protein of this pathway, and thus links the G-protein to the downstream MAPK cascade (Cheng et al., 2015).

In human COS-7 and HEK293 cells, RACK1 interacts with the MAP3K MTK1, which regulates apoptosis via its downstream targets p38 and JNK in response to different types of stress (Arimoto et al., 2008). Activation of MTK1 requires the formation of a MTK1-homodimer, which leads to MTK1 autophosphorylation. In the absence of stress, RACK1 keeps MTK1 in a dimeric, however, inactive form. Thereby, it facilitates the subsequent activation as soon as stress conditions appear (Arimoto et al., 2008). As mentioned above, distinct stresses like arsenite or hypoxia cause RACK1 to move into stress granules (Arimoto et al., 2008). Thus, these stress conditions impede MTK1 activation due to RACK1-MTK1 dissociation (Arimoto et al., 2008).

The extracellular signal-regulated kinase (ERK) is a MAPK that responds to extracellular stimuli, such as integrin-mediated signals. RACK1 was identified as interaction partner not only for ERK1 and ERK2, but also for the corresponding MAP2Ks Raf-1 and B-Raf and the

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(Vomastek et al., 2007). Accordingly, the absence of RACK1 results in decreased pathway activation in response to integrin in REF52 fibroblasts (Vomastek et al., 2007). Moreover, ERK signaling controls the transcription and stability of c-Jun, a regulator of RACK1 transcription, which suggests a connection to the JNK signaling pathway (Lopez-Bergami et al., 2007).

RACK1 is indeed engaged in the JNK pathway, a stress-activated MAPK pathway. It interacts with the JNK-specific MAP2K MKK7 in human hepatocellular carcinoma cells (Guo et al., 2013). This interaction increases the phosphorylation of both MKK7 and the MAPK JNK and facilitates the interaction between MKK7 and the corresponding MAP3K (Guo et al., 2013).

RACK1 was also shown to interact with JNK itself (López-Bergami et al., 2005). Furthermore, RACK1 represents a functional and physical link between JNK and PKC signaling as it functions as molecular bridge between both kinases and thus enables the phosphorylation of JNK at Ser129 through PKC. This modification seems to enhance in turn the phosphorylation of JNK by its two MAP2Ks MKK7 and MKK4 (López-Bergami et al., 2005). The other way around, sufficient MKK4/MKK7 protein levels are required to allow for PKC-dependent JNK phosphorylation (López-Bergami et al., 2005).