Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway

Abstract

Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinaseactivated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.ERK3 and ERK4 define a distinct subfamily of atypical MAP kinases that display structural and functional differences with conventional MAP kinases (1). First, they possess a single phospho-acceptor site (Ser-Glu-Gly) in their activation loop instead of the classical dual phosphorylation Thr-Xxx-Tyr motif (2, 3). Accordingly, they are not phosphorylated and activated by dual-specificity MAP kinase kinase family members (4, 5). Second, ERK3 and ERK4 bear the sequence Ser-Pro-Arg instead of Ala-Pro-Glu in subdomain VIII of the kinase domain, being the only kinases in the human kinome to have an arginine at this position. Third, contrary to conventional MAP kinases like ERK1/ERK2, which are multifunctional Ser/Thr kinases capable of phosphorylating a vast array of substrates, ERK3 and ERK4 appear to have a very narrow substrate specificity (6). Their only known physiological substrate is the protein kinase MK5 5 (7-10). The regulation of ERK3 and ERK4 activity remains poorly understood. We and others have shown that the serine residue within the activation loop of ERK3/ERK4 (Ser-189/Ser-186) is phosphorylated in vivo (6, 11-13). Catalytically inactive forms of the kinases are similarly phosphorylated on this motif, indicating that activation loop phosphorylation is mediated by an upstream cellular kinase (12, 13). Phosphorylation of Ser-189/Ser-186 leads to enzymatic activation of ERK3/ERK4 and is required for binding to and for cytoplasmic relocalization of the substrate MK5 (12, 13). Recently, we have show...

FSP-1 Silencing in Bone Marrow Cells Suppresses Neointima Formation in Vein Graft

“…Furthermore, we found that Rac1 can regulate FSP-1-induced MEK5/ERK5 phosphorylation and VSMC proliferation ( Figure 7G and 7H). Similar responses have been reported that Rac1 involved in regulating MAPK activation through p21-activated kinases 36 or JNK/AP1 pathway. 37 In contrast, FSP-1-stimulated Rac1 activation could not be inhibited by negative dominant MEK5, suggesting that Rac1 acts as an upstream of ERK5.…”

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades

“…Some of the atypical MAPKs have to be phosphorylated on a single serine residue by IPAKs for elicitation [5]. Other representatives of atypical MAPKs, such as ERK7/8 gain their activity by intramolecular autophosphorylation on both phosphoacceptor sites of the TXY motif [8,9].…”

“…Isolation of protoplast and polyethylene glycol-mediated protoplast transformation were carried out as described by Anthony et al [20]. We used 5 μg plasmid DNAs to transform 2x10 5 protoplasts, and then cells were cultured for 16 h in dark at room temperature before harvesting. In case of myc-GOF-AtMKK co-transformation, 5:1 ratio was applied; 5 μg of AtMPK9-HA coding vector and 1 μg of different myc-GOF-MKK constructs.…”

“…Both kinases lack the tyrosine phosphoacceptor site of the conserved TXY motif, but contain a SEG amino acid triplet and phosphorylation of the single serine residue within this motif is sufficient for their activation [4]. ERK3/4 cannot be phosphorylated and activated by any of the MKKs but instead they are phosphorylated by a group of I p21-activated kinases (I PAKs) which act as upstream activators for these MAPKs [5]. ERK7/8 resemble to ERK1/2 and ERK5 in having a TEY phosphoacceptor motif in the activation loop [6,7].…”

ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion

“…ERK3 was recently shown to be activated by group I p21-activated kinases (PAKs) that include PAK1, PAK2, and PAK3 (29,30). Group I PAKs are important regulators for cell migration and invasion.…”