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ONCOGENOMICS Logs, Mushroom Bodies Tiny (MBT) and C45B11.1, the Importance of P21 Activated Kinase (PAK) Family Respectively (Dan Et Al., 2001)

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ONCOGENOMICS Logs, Mushroom Bodies Tiny (MBT) and C45B11.1, the Importance of P21 Activated Kinase (PAK) Family Respectively (Dan Et Al., 2001) Oncogene (2002) 21, 3939 ± 3948 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain Akhilesh Pandey*,1,4, Ippeita Dan*,2,4, Troels Z Kristiansen1, Norinobu M Watanabe2, Jesper Voldby1, Eriko Kajikawa2, Roya Khosravi-Far3, Blagoy Blagoev1 and Matthias Mann*,1 1Center for Experimental Bioinformatics, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark; 2Kusumi Membrane Organizer Project, ERATO, JST, Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602 Japan; 3Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical Center, Boston, Massachusetts, MA 02115, USA The p21-activated kinase (PAK) family of protein small GTP-binding proteins, Rac and CDC42, PAK kinases has recently attracted considerable attention as family kinases (PAKs) participate in various facets of an eector of Rho family of small G proteins and as an cellular events. In addition to modulating the organiza- upstream regulator of MAPK signalling pathways during tion of the actin cytoskeleton to control cell morphol- cellular events such as re-arrangement of the cytoskele- ogy and motility, PAKs activate mitogen-activated ton and apoptosis. We have cloned a novel human PAK protein kinase (MAPK) signalling pathways to aect family kinase that has been designated as PAK5. PAK5 gene expression (Sells and Cherno, 1997; Bagrodia contains a CDC42/Rac1 interactive binding (CRIB) and Cerione, 1999). More recently, PAKs have also motif at the N-terminus and a Ste20-like kinase domain been implicated in apoptosis and have been shown to at the C-terminus. PAK5 is structurally most related to possess either pro-apoptotic or anti-apoptotic proper- PAK4 and PAK6 to make up the PAK-II subfamily. We ties in dierent studies (Rudel and Bokoch, 1997; have shown that PAK5 preferentially binds to CDC42 in Schurmann et al., 2000, Tang et al., 2000, Jakobi et al., the presence of GTP and that CRIB motif is essential 2001, Gnesutta et al., 2001). for this interaction. PAK5 is a functional protein kinase The PAK family together with the GCK (germinal but unlike PAK-I family kinases (PAK1, 2, and 3), the centre kinase) family comprises the Ste20 (Sterile20) kinase activity of PAK5 does not seem to require the group of kinases in higher eukaryotes. They all share binding of CDC42. Overexpression of PAK5 activates an evolutionarily conserved Ste20-like kinase domain. the JNK kinase pathway but not p38 or ERK pathways. However, the PAK family members are distinguished PAK5 transcript is predominantly expressed in brain as from GCK family kinases in that they have a kinase revealed by Northern blot and in situ hybridization. The domain at their C terminus instead of at the N expression pattern of PAK5 is distinct from that of terminus. A total of six PAK family members has been PAK4 and PAK6, suggesting a functional division among identi®ed thus far in humans, including PAK5 reported PAK-II subfamily kinases based on dierential tissue in this paper. They have been sequentially numbered distribution. from 1 to 6. Recent extensive analysis based on human, Oncogene (2002) 21, 3939 ± 3948. DOI: 10.1038/sj/ Drosophila and C. elegans genomic sequences has onc/1205478 revealed that the PAK family can be classi®ed into two distinct subfamilies. The PAK-I subfamily consists Keywords: p21-activated kinase; Ste20; CDC42; JNK; of three mammalian kinases PAK1, 2 and 3 together brain with their Drosophila and C. elegans orthologs, DPAK and CePAK (C09B8.7), while PAK-II subfamily consists of three mammalian kinases PAK4, 5 and 6 Introduction together with their Drosophila and C. elegans ortho- ONCOGENOMICS logs, mushroom bodies tiny (MBT) and C45B11.1, The importance of p21 activated kinase (PAK) family respectively (Dan et al., 2001). of kinases has become increasingly realized in cellular All PAK family kinases share two common signalling pathways. As eectors of the Rho family of structural features: a kinase domain at the C-terminus and a CDC42/Rac-interactive-binding (CRIB) domain in the middle of the N-terminal non-catalytic region. The PAK-I subfamily kinases have some additional *Correspondence: A Pandey, I Dan or M Mann; characteristic motifs. The ®rst is an auto-inhibitory E-mail: [email protected] or [email protected] or domain located downstream of the CRIB domain [email protected] which partially overlaps with the CRIB domain itself. 4The ®rst two authors contributed equally to this work Received 2 January 2002; revised 13 March 2002; accepted 13 PAK-Is are inactivated by intramolecular interaction of March 2002 the auto-inhibitory domain with the kinase domain. A novel p21-activated kinase, PAK5 A Pandey et al 3940 The binding of CDC42 or Rac to the CRIB domain ubiquitously expressed in most tissues, while PAK6 is disrupts this interaction, leading to the activation of speci®cally expressed in testes and prostate (Abo et al., the kinase activity. The second is a Cool/Pix-binding 1998; Yang et al., 2001). In Ste20 group kinases, motif, which is crucial for the recruitment of PAK-Is to functional division among subfamily members is often focal adhesion complexes upon stimulation by CDC42/ established by dierential expression patterns. There- Rac-1. In addition, proline rich motifs implicated in fore, it has been anticipated that there exists a human the binding of SH3 domain-containing adapter pro- PAK-II kinase that may be speci®cally expressed in teins such as Nck, are present at the extreme N- brain. terminal region, while the C-terminal region is In search of the missing member of the PAK-II implicated as a binding site for the b subunit of subfamily, we have cloned a novel kinase of the PAK- heterotrimeric G-proteins. PAK-Is are highly homo- II subfamily, PAK5. In this paper, we show that PAK5 logous to the yeast Ste20p, sharing both CRIB and is predominantly expressed in brain. PAK5 is a protein auto-inhibitory domains (Sells and Cherno, 1997; kinase that activates JNK (Jun N-terminus kinase) but Bagrodia and Cerione, 1999; Dan et al., 2001). These not p38 or ERK (extracellular signal-regulated kinase). kinases are so highly conserved that PAK3 from PAK5 interacts with CDC42 and this interaction is humans is able to complement Ste20p defects in the mediated by the CRIB motif in the N-terminus. Unlike budding yeast (Bagrodia et al., 1995a). the case with PAK-Is, the binding of CDC42 is not Subfamily II PAK members have a CRIB domain necessary for the kinase activity of PAK5. without a recognizable auto-inhibitory domain, and they lack binding motifs for Cool/Pix, Nck and Gb (Sells and Cherno, 1997; Bagrodia and Cerione, 1999; Dan et al., 2001). Function of PAK-IIs is only Results and discussion beginning to be understood. PAK4, the only PAK-II member characterized in detail thus far, is involved in Cloning of PAK5 reorganization of the actin cytoskeleton in a manner that is distinct from that of PAK-Is. Unlike PAK-Is, The existence of the PAK5 gene in the human genome the kinase activity of PAK4 is not dependent on Rho was ®rst pointed out in our systematic phylogenetic family GTPases. CDC42 can bind to PAK4 but is analysis of the PAK family of kinases (Dan et al., involved in the translocation of PAK4 to the Golgi 2001). While the PAK-I subfamily of kinases consists apparatus, rather than its activation (Abo et al., 1998). of three paralogs in the human genome, only one PAK4 has been shown to induce ®lopodia formation member, PAK4, had been found for the PAK-II through the re-arrangement of the cytoskeleton, but its subfamily. In searching for additional molecules related regulatory mechanism remains uncertain. Recently, X- to PAK-4, we performed RT ± PCR (reverse transcrip- PAK5, a novel PAK-II kinase in Xenopus laevis, was tion polymerase chain reaction) using degenerate demonstrated to bind to actin and microtuble networks primers. The design of these primers was based on a (Cau et al., 2001). Because X-PAK5 is most related to conserved CRIB motif and kinase domain of PAK4 human PAK4 and more distantly to PAK5 and PAK6, (Abo et al., 1998). From total RNA of adult human it is likely to be the Xenopus laevis ortholog of human brain, we ®rst obtained a 1.7 kb cDNA fragment. A PAK4. It has been suggested that X-PAK5 dissociates sequence analysis of the fragment revealed that it from microtubules upon catalytic activation, and encoded a novel PAK4-like kinase. Since this clone did subsequent binding to CDC42/Rac1 GTPases causes not contain the entire open reading frame, 5' and 3' it to translocate to actin cytoskeleton such as stress RACE (rapid ampli®cation of cDNA ends) experi- ®bres and lamellipodia (Cau et al., 2001). An entirely ments were performed in order to obtain a longer novel functional role has been assigned to recently insert. The longest clone we obtained harbors a cloned PAK6 despite its structural similarity to other 4612 bp insert containing an open reading frame of PAK-IIs. PAK6 is preferentially expressed in testis and 719 amino acids with the initiator methionine following binds the androgen receptor. In response to androgen, a 380 bp-long 5' UTR (untranslated region) with it translocates to the nucleus resulting in repression of multiple in-frame stop codons (Figure 1a). Another androgen receptor-mediated transcription (Yang et al., cDNA entry in the database (KIAA1264; GenBank 2001). accession no. AB033090) (Nagase et al., 1999) contains Drosophila ortholog of PAK-IIs, MBT, is suggested an additional 34 amino acid residues as compared to to play integral roles in neuronal morphogenesis since our protein sequence with the start codon annotated as its de®ciency results in severe defects in central brain `not identi®ed.' We have reported earlier that com- structures in adult ¯ies.
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