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PAK lies downstream of Chp signaling and active kinase co-localizes with E-cadh. (A) Characterization of the phospho-PAK (pPAK) antibody. Alignment of auto-inhibitory domain (AID) of human (Hs) and zebrafish (Zf) PAKs: HsPak1, NP_001122092.1; HsPak2, NP_002568.2; HsPak3, NP_002569.1; ZfPak1, NP_958485.1; ZfPak2a, NP_001002717; and ZfPak2b, NP_001020627.1. Dark and light grey shaded sequences represent identical and conserved amino acids; the serine residue highlighted in red is the phosphorylated site recognized by the rabbit anti-pPAK144 antibody. Further characterization of this new antibody will be presented elsewhere; it is ∼10 times more sensitive than the anti-PAK1 pSer199 described previously in zebrafish [85]. Anti-pS144PAK1 recognizes all zebrafish PAK isoforms as assessed by synthetic phospho-peptides representing zebrafish PAK sequences. The peptides were synthesized in situ on cellulose (Jerinini) with a 3 amino-acid linker at the C-terminal end, and N-terminally amidated. The filter was blocked with BSA, and probed with anti-pPAK and HRP-anti-rabbit IgG antibodies both at 1:2000 (30 min each, with 3x10 min washes). Human PAK3 (pS139) is shown as a positive control. (B) Constitutively active Chp (G38V) can activate PAK1. Active Cdc42(G12V), Chp(G38V) and RhoUa(Q104L) were cloned in the mammalian expression vector pXJ-Flag (with CMV promoter) and co-expressed with HA-tagged PAK1 in COS-7 cells. The activation of PAK1 is indicated by an upshift in the PAK1 band and by PAK1 phosphorylation on Ser144. (C) Activated pPAK1 was detected at the centrosome of mitotic cell (as previously reported in mammalian cell culture) and can be found at the cell junctions of envelope cells (EVL), but not on the junctions of deeper cells. (D) E-cadh colocalizes with pPAK1 at the AJs of the EVL but not in the cytoplasmic puncta. Both were reduced at the AJs of the EVL in Chp morphants. Junctional pPAK signal is reduced in the envelope cells in PIX morphants. Typical stainings of control and PIX morphant embryos. Both images represent a stack of 3 confocal images, collected under the same laser and gain settings, and at equivalent positions in the embryo. White arrows represent junctional pPAK1. Scale bars represent 20 μm.
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