Substrates enhance autophosphorylation and activation of p21‐activated protein kinase γ‐PAK in the absence of activation loop phosphorylation

The class I p21-activated kinases (Pak1-3) regulate many essential biological processes, including cytoskeletal rearrangement, cell cycle progression, apoptosis, and cellular transformation. Although many Pak substrates, including elements of MAPK signaling cascades, have been identified, it is likely that additional substrates remain to be discovered. Identification of such substrates, and determination of the consequences of their phosphorylation, is essential for a better understanding of class I Pak activity. To identify novel class I Pak substrates, we used recombinant Pak2 to screen high density protein microarrays. This approach identified the atypical MAPK Erk3 as a potential Pak2 substrate. Solution-based in vitro kinase assays using recombinant Erk3 confirmed the protein microarray results, and phospho-specific antisera identified serine 189, within the Erk3 activation loop, as a site directly phosphorylated by Pak2 in vitro. Erk3 protein is known to shuttle between the cytoplasm and the nucleus, and we showed that selective inhibition of class I Pak kinase activity in cells promoted increased nuclear accumulation of Erk3. Pak inhibition in cells additionally reduced the extent of Ser 189 phosphorylation and inhibited the formation of Erk3-Prak complexes. Collectively, our results identify the Erk3 protein as a novel class I Pak substrate and further suggest a role for Pak kinase activity in atypical MAPK signaling.The class I p21-activated kinases (Pak1-3) are established effectors of the small GTPases Rac1 and Cdc42 (1). Although initially discovered as regulators of the actin cytoskeleton, they have subsequently been implicated in a variety of different signaling pathways, including those that control proliferation, apoptosis, and transformation (2, 3). Not surprisingly, misregulated Pak 2 kinase activity is associated with a variety of different pathological conditions (4 -6). As Pak kinase activity contributes to several different signaling pathways, a better understanding of the specific role for Paks in any biological response is greatly aided by the identification of the protein(s) they phosphorylate and the consequences of these phosphorylations. Although the current list of Pak kinases substrates is quite large (3, 7), we are particularly interested in how Pak kinases influence MAPK signaling cascades.MAPK signaling pathways are among the most evolutionarily conserved signal transduction pathways and are critical for many biological responses (8). Various cellular stimuli lead to the enzymatic activation of a family of serine/threonine kinases known as MAP3Ks. Activated MAP3Ks phosphorylate and activate specific MAP2Ks. MAP2Ks are dual specificity kinases that phosphorylate threonine and tyrosine residues within the activation loop of the MAPKs (the conserved TXY motif) to activate them; phosphorylation of both activation loop residues is required for MAPK activation. Activated MAPKs then transphosphorylate a variety of different proteins, including structural proteins, enzymes, and transcrip...

Section: High Density Protein Microarrays Identify Erk3 As a Potentialsupporting

confidence: 79%

Identification of the Atypical MAPK Erk3 as a Novel Substrate for p21-activated Kinase (Pak) Activity

Mota-Peynado

Chernoff

Beeser

2011

“…Recently, a phosphorylation state-specific antibody directed against Thr 402 of PAK2, analogous to Thr 423 of PAK1 (27), was found to cross-react with endogenous ␣-PAK (PAK1) and MST kinases in osmotically or chemically stressed neutrophils (28). We utilized this sequence conservation to prove phosphorylation at Thr 183 of MST1 in the wild-type kinase.…”

Section: Discussionmentioning

confidence: 99%

Mapping of MST1 Kinase Sites of Phosphorylation

Glantschnig

Rodan

Reszka

2002

165

171

MST1 is a member of theIn mammalian cells, Sterile-20 (Ste20)-related kinases participate in the regulation of the cytoskeleton that controls cell morphology and motility, and in the regulation of apoptosis (1). These kinases share a conserved catalytic (kinase) domain at the amino terminus and a C-terminal regulatory region of great structural diversity, which interacts with signaling molecules that regulate the cytoskeleton. Currently, four closely related MST kinases have been described (5-9). Most Ste20 group kinases activate mitogen-activated protein kinase (MAPK) 1 cascades in the signaling pathways between the cellular membrane and the nuclear compartment (2). In yeast, the mating pheromone receptor Ste20p phosphorylates and activates a MAPK kinase kinase, Ste11p, raising the possibility that mammalian homologs of Ste20p (e.g. MST1 kinase) also function as MAPK kinase kinase kinases (3, 4). MST1 was shown to act upstream of MAPK kinases that regulate p38 and JNK activities, probably acting via the MAPK kinase kinase MEKK1 (10).There is substantial evidence that MST1 promotes apoptosis, although its role in this process may vary in different cell types. Overexpression of MST1 can induce apoptosis and nuclear condensation in BJAB, 293T and COS-1 cells (4, 14, 15), whereas MST1 promotes nuclear condensation without apparent chromosomal cleavage in HeLa cells (13).A consistent feature of MST1 in all tested cell types is its proteolytic cleavage by caspase, in response to apoptotic stimuli, to a 34 -36-kDa product (hereafter referred to as 36-kDa MST1). Cleavage increases MST1 kinase activity severalfold (4, 16 -19) and influences its subcellular localization (13, 15). Recently, a second caspase cleavage site was identified in the human MST1 sequence that is absent in mouse MST1 and in MST2 from several species (10). Mutation of this site has no impact on accumulation of the 36-kDa species.Expression of a kinase-dead MST1 mutant (K59R) can partially or fully suppress apoptosis or chromatin condensation in HL-60 and 293T cells treated with chemical apoptotic stimuli (15,18). This protective effect is associated with suppression of both JNK activity and DNA fragmentation. However, the K59R mutant fails to suppress apoptosis in BJAB and HeLa cells treated with Fas ligand or TNF-␣ (4, 16). The basis for the cell type-and stimulus-specific differences in MST function has not yet been elucidated.Interestingly, like PAK proteins (11, 12), MST1 (full-length or cleaved) can have a profound effect on cell shape (cell rounding and detachment) independent of caspase activation and prior to nuclear condensation (13). This action and the high basal activity of MST1 kinase suggest a possible function unrelated to apoptosis.Stress-inducing agents such as staurosporine and sodium arsenite (9) can increase MST1 kinase activity; however, no physiological activator of MST1 has been identified, and little is known about the endogenous activation of MST1. Recently, it was proposed that in addition to caspase cleavage, MST1 phosphor...

“…Caspase activation has not been demonstrated for any other PAK isoform and among the PAK family appears to be a unique characteristic for PAK-2. Activation by both Cdc42(GTP) and caspases requires autophosphorylation at Thr-402 of PAK-2 (15,17).…”

mentioning

confidence: 99%

Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Jakobi

McCarthy

Koeppel

et al. 2003

Self Cite

p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (␥-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor ␣ or growth factor withdrawal, PAK-2 is activated as a fulllength enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of fulllength PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.