Group I and II mammalian PAKs have different modes of activation by Cdc42 - PubMed (original) (raw)

Group I and II mammalian PAKs have different modes of activation by Cdc42

Yohendran Baskaran et al. EMBO Rep. 2012.

Abstract

p21-activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1-like (group I) or PAK4-like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified auto-inhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A-loop phosphorylation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

PAK4 activity is independent of Ser 474 phosphorylation. (A) Schematic of the PAK4 gene in chromosome 19q13.2 and the exon organization of human PAK4. The PAK4b splice isoform lacks exon 4: identified domains include the nuclear localization signal (NLS, black), Cdc42–Rac interaction/binding (CRIB, blue) and catalytic domain (red). Other conserved but undefined regions in group II PAKs are in green. The number of human EST (expressed sequence tags) in the NCBI database is indicated. (B) Reverse transcriptase–PCR analysis (RT–PCR, 30 cycles) of mRNA isolated from HeLa, U2OS and mouse B16 cell lines. The position of the PAK4a and PAK4b cDNA products are marked. (C) Western blots (WBs) of cell lysates derived cell lines (40 μg) as indicated were probed with affinity-purified antibodies raised against PAK4(1–280) or a pSer 474 13mer peptide. The PAK4 and Cdc42 targeted short interfering RNAs (siRNAs) have been described previously [12]. The asterisk marks a 72-kDa nonspecific band. (D) Alignment of sequences surrounding known PAK4 substrate sites. (E) Flag–PAK4 constructs were immunoprecipitated (IP) and assayed in vitro with [32-P]ATP with the Raf1 S338/9 peptide substrate (see Methods). Autoradiograph on the top panel is a 2-h exposure. The A-loop phosphorylation was probed using CS#3241. The red box highlights that PAK4–Cat does not undergo auto-phosphorylation. CA, constitutively active, PAK4(S445N); Cat, PAK4(286–591); GST, glutathione _S_-transferase; HA, haemagglutinin; KD, kinase dead, PAK4(K350M); PAK, p21-activated kinase; PAK4c lacks residues 121–285, under accession number AAH02921.1.

Figure 2

Figure 2

Identification of a conserved PAK4 auto-inhibitory domain (AID). (A) An alignment of the AID (orange bar) comparing group I PAKs with those from metazoan PAK4 and fungal and protozoan PAKs. The three α-helices in the PAK1 AID are marked (red bars). The A-loop binding motif (KYMS/T, pink box) is not found in fungal or protozoan PAKs. Proline residues preceding helixes 1 and 3 are marked in grey. Conserved residues involved in Cdc42 binding are in blue. Hydrophobic AID residues are highlighted in yellow, while those conserved only among group II PAKs are in green. The position of the PAK4 AID mutations tested in this study are indicated by asterisk marks (potential phosphorylation sites) or underlined in green (R48, R49). (B) Flag–tagged PAK4/5 and 6 expressed in COS7 were analysed for activation loop phosphorylation. (C) Schematic of a minimal synthetic construct PAK4(s) comprising residues PAK4(1–68) joined to PAK4(286–591) with a 10-residue linker. (D) The various Flag–PAK4 were used for in vitro kinase assays performed as described in Fig 1. The A-loop phosphorylation was probed using CS#3241. Cat, PAK4(286–591); CRIB, Cdc42–Rac interaction/binding; GST, glutathione _S_-transferase; IP, immunoprecipitated; KD, kinase dead, PAK, p21-activated kinase; PAK4a(K350M); WB, western blot; WT, wild type; Δ20, PAK4a(21–591) and Δ50, PAK4a(51–591).

Figure 3

Figure 3

Cdc42.GTP directly activates PAK4. (A) A schematic of the pull-down protocol from COS7 cell lysates is shown. The PAK4 proteins were recovered as indicated, and assayed for activity towards glutathione _S_-transferase (GST)–Raf13 (20 μg) in the presence of 10 μM [γ32-P] ATP. The pS474 was probed using CS#3241. (B) Schematic of the active Cdc42–Gly6–PAK4 chimaera (Flag-C–PAK4). (C) The relative in vitro kinase activity of purified PAK4 tested in three independent experiments was calculated after subtracting PAK4 KD (that is, background value) and setting the Flag–PAK4 value to 1. The bars represent the standard error for each measurement. (D) The efficiency of green fluorescent protein–GEFH1 binding to various Flag–PAK4 constructs as indicated was tested by coimmunoprecipitation. CA, constitutively active; Cat, PAK4(286–591); CRIB, Cdc42–Rac interaction/binding; GFP, green fluorescent protein; GST, glutathione _S_-transferase; HA, haemagglutinin; IP, immunoprecipitated; KD, kinase dead; PAK, p21-activated kinase; WB, western blot; WT, wild type; Δ50=PAK4a(51–591).

Figure 4

Figure 4

An auto-inhibitory domain (AID) active mutant has reduced binding to Cdc42. (A) Constructs of wild-type or mutant PAK4a as indicated were expressed in Cos7 cells, immunoprecipitated and assayed for activity in vitro. WT, wild type; AID**, RR48/49AE, T60E; Cat, PAK4(286–591). (B) PAK4a, PAK4b and the active PAK4a–AID** were immunoprecipitated and tested for the presence of coexpressed HA–Cdc42V12. (C) HeLa cells were serum-starved (16 h), treated with HGF and harvested at the times shown. Triton X-100 soluble cell lysates (30 μg/lane) were probed with new antibodies directed to PAK4, and PAK4 pS474, or ERK1/2 and pERK1/2 (Cell Signaling). (D) This model summarizes our findings regarding the alternate activation mechanisms for group I and II PAKs. Cdc42 binding in both cases results in relief from auto-inhibition, but through different underlying mechanisms. In the case of PAK1, this requires new phosphorylation of Ser 144 and Thr 423. For simplicity we do not show the PAK1 as a dimer. In contrast, PAK4 is constitutively phosphorylated on Ser 474, and requires Cdc42.GTP to induce and sustain an active kinase conformation that allows substrate interaction. HA, haemagglutinin; HGF, hepatocyte growth factor; IB, immunoblot; IP, immunoprecipitated; PAK, p21-activated kinase; WB, western blot; WT, wild type.

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