P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1Δ15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1Δ15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition (IPA-3, FRAX597) or depletion (siRNA) on cell-surface adhesion were monitored by real-time microimpedance measurement. Both PAK1Δ15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using coimmunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterocomplexes were also detected. Interaction of PAK1Δ15 or PAK2 with PAK1-full was associated with extensive PAK1Δ15/PAK2 cleavage. The impedance measurements indicate, that PAK2 depletion slows down cell attachment to a surface, and that PAK1-full is involved in cell spreading. Altogether, our data suggest a complex interplay among different PAK group I members, which have non-redundant functions.

Department of Proteomics Institute of Hematology and Blood Transfusion U Nemocnice 1 128 20 Prague Czech Republic

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Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994;367:40–46. doi: 10.1038/367040a0. PubMed DOI

Knaus UG, Morris S, Dong HJ, Chernoff J, Bokoch GM. Regulation of human leukocyte p21-activated kinases through G protein–coupled receptors. Science. 1995;269:221–223. doi: 10.1126/science.7618083. PubMed DOI

Bokoch GM. Biology of the p21-activated kinases. Annu. Rev. Biochem. 2003;72:743–781. doi: 10.1146/annurev.biochem.72.121801.161742. PubMed DOI

Molli PR, Li DQ, Murray BW, Rayala SK, Kumar R. PAK signaling in oncogenesis. Oncogene. 2009;28:2545–2555. doi: 10.1038/onc.2009.119. PubMed DOI PMC

Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer. Nat. Rev. Cancer. 2014;14:13–25. doi: 10.1038/nrc3645. PubMed DOI PMC

Singh RR, Song C, Yang Z, Kumar R. Nuclear localization and chromatin targets of p21-activated kinase 1. J. Biol. Chem. 2005;280:18130–18137. doi: 10.1074/jbc.M412607200. PubMed DOI

Kumar R, Li DQ. PAKs in Human Cancer Progression: From Inception to Cancer Therapeutic to Future Oncobiology. Adv. Cancer Res. 2016;130:137–209. doi: 10.1016/bs.acr.2016.01.002. PubMed DOI

Coniglio SJ, Zavarella S, Symons MH. Pak1 and Pak2 mediate tumor cell invasion through distinct signaling mechanisms. Mol. Cell. Biol. 2008;28:4162–4172. doi: 10.1128/MCB.01532-07. PubMed DOI PMC

Arias-Romero LE, Chernoff J. A tale of two Paks. Biol. Cell. 2008;100:97–108. doi: 10.1042/BC20070109. PubMed DOI

Manser E, et al. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell. 1998;1:183–192. doi: 10.1016/S1097-2765(00)80019-2. PubMed DOI

Obermeier A, et al. PAK promotes morphological changes by acting upstream of Rac. EMBO J. 1998;17:4328–4339. doi: 10.1093/emboj/17.15.4328. PubMed DOI PMC

Lei M, et al. Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch. Cell. 2000;102:387–397. doi: 10.1016/S0092-8674(00)00043-X. PubMed DOI

Zenke FT, King CC, Bohl BP, Bokoch GM. Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity. J. Biol. Chem. 1999;274:32565–32573. doi: 10.1074/jbc.274.46.32565. PubMed DOI

Parrini MC, Lei M, Harrison SC, Mayer BJ. Pak1 kinase homodimers are autoinhibited in trans and dissociated upon activation by Cdc42 and Rac1. Mol. Cell. 2002;9:73–83. doi: 10.1016/S1097-2765(01)00428-2. PubMed DOI

Parrini MC. Untangling the complexity of PAK1 dynamics: The future challenge. Cell. Logist. 2012;2:78–83. doi: 10.4161/cl.19817. PubMed DOI PMC

Shin YJ, Kim YB, Kim JH. Protein kinase CK2 phosphorylates and activates p21-activated kinase 1. Mol. Biol. Cell. 2013;24:2990–2999. doi: 10.1091/mbc.e13-04-0204. PubMed DOI PMC

Harms FL, et al. Activating Mutations in PAK1, Encoding p21-Activated Kinase 1, Cause a Neurodevelopmental Disorder. Am. J. Hum. Genet. 2018;103:579–591. doi: 10.1016/j.ajhg.2018.09.005. PubMed DOI PMC

Combeau G, et al. The p21-activated kinase PAK3 forms heterodimers with PAK1 in brain implementing trans-regulation of PAK3 activity. J. Biol. Chem. 2012;287:30084–30096. doi: 10.1074/jbc.M112.355073. PubMed DOI PMC

Puto LA, Pestonjamasp K, King CC, Bokoch GM. p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling. J. Biol. Chem. 2003;278:9388–9393. doi: 10.1074/jbc.M208414200. PubMed DOI

Zhou GL, et al. Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration. Mol. Cell. Biol. 2003;23:8058–8069. doi: 10.1128/MCB.23.22.8058-8069.2003. PubMed DOI PMC

Tao J, Oladimeji P, Rider L, Diakonova M. PAK1-Nck regulates cyclin D1 promoter activity in response to prolactin. Mol. Endocrinol. 2011;25:1565–1578. doi: 10.1210/me.2011-0062. PubMed DOI PMC

Hammer A, Oladimeji P, De Las Casas LE, Diakonova M. Phosphorylation of tyrosine 285 of PAK1 facilitates betaPIX/GIT1 binding and adhesion turnover. FASEB J. 2015;29:943–959. doi: 10.1096/fj.14-259366. PubMed DOI PMC

Banerjee M, Worth D, Prowse DM, Nikolic M. Pak1 phosphorylation on t212 affects microtubules in cells undergoing mitosis. Curr. Biol. 2002;12:1233–1239. doi: 10.1016/S0960-9822(02)00956-9. PubMed DOI

King CC, et al. p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1) J. Biol. Chem. 2000;275:41201–41209. doi: 10.1074/jbc.M006553200. PubMed DOI

Rudel T, Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science. 1997;276:1571–1574. PubMed

Hsu YH, Johnson DA, Traugh JA. Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange. J. Biol. Chem. 2008;283:36397–36405. doi: 10.1074/jbc.M805581200. PubMed DOI PMC

Wilkes MC, Murphy SJ, Garamszegi N, Leof EB. Cell-type-specific activation of PAK2 by transforming growth factor beta independent of Smad2 and Smad3. Mol. Cell. Biol. 2003;23:8878–8889. doi: 10.1128/MCB.23.23.8878-8889.2003. PubMed DOI PMC

Chan WH, Yu JS, Yang SD. PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: evidence for the involvement of oxidative stress. J. Cell. Physiol. 1999;178:397–408. doi: 10.1002/(SICI)1097-4652(199903)178:3<397::AID-JCP14>3.0.CO;2-2. PubMed DOI

Tang TK, et al. Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells. J. Cell. Biochem. 1998;70:442–454. doi: 10.1002/(SICI)1097-4644(19980915)70:4<442::AID-JCB2>3.0.CO;2-J. PubMed DOI

Roig J, Traugh JA. Cytostatic p21 G protein-activated protein kinase gamma-PAK. Vitam. Horm. 2001;62:167–198. doi: 10.1016/S0083-6729(01)62004-1. PubMed DOI

Marlin JW, Eaton A, Montano GT, Chang YW, Jakobi R. Elevated p21-activated kinase 2 activity results in anchorage-independent growth and resistance to anticancer drug-induced cell death. Neoplasia. 2009;11:286–297. doi: 10.1593/neo.81446. PubMed DOI PMC

Marlin JW, et al. Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34. Mamm. Genome. 2011;22:306–317. doi: 10.1007/s00335-011-9326-6. PubMed DOI

Li X, et al. Phosphorylation of caspase-7 by p21-activated protein kinase (PAK) 2 inhibits chemotherapeutic drug-induced apoptosis of breast cancer cell lines. J. Biol. Chem. 2011;286:22291–22299. doi: 10.1074/jbc.M111.236596. PubMed DOI PMC

Ye DZ, Field J. PAK signaling in cancer. Cell. Logist. 2012;2:105–116. doi: 10.4161/cl.21882. PubMed DOI PMC

Huynh N, et al. Depletion of p21-activated kinase 1 up-regulates the immune system of APC(14/+) mice and inhibits intestinal tumorigenesis. BMC Cancer. 2017;17:431-017-3432-0. doi: 10.1186/s12885-017-3432-0. PubMed DOI PMC

Pandolfi A, et al. PAK1 is a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome. Blood. 2015;126:1118–1127. doi: 10.1182/blood-2014-12-618801. PubMed DOI PMC

Semenova G, Chernoff J. Targeting PAK1. Biochem. Soc. Trans. 2017;45:79–88. doi: 10.1042/BST20160134. PubMed DOI PMC

Maruta H, Ahn MR. From bench (laboratory) to bed (hospital/home): How to explore effective natural and synthetic PAK1-blockers/longevity-promoters for cancer therapy. Eur. J. Med. Chem. 2017;142:229–243. doi: 10.1016/j.ejmech.2017.07.043. PubMed DOI

Zandvakili I, Lin Y, Morris JC, Zheng Y. Rho GTPases: Anti- or pro-neoplastic targets? Oncogene. 2017;36:3213–3222. doi: 10.1038/onc.2016.473. PubMed DOI PMC

Kumar R, Sanawar R, Li X, Li F. Structure, biochemistry, and biology of PAK kinases. Gene. 2017;605:20–31. doi: 10.1016/j.gene.2016.12.014. PubMed DOI PMC

Liu X, et al. JMJD6 promotes melanoma carcinogenesis through regulation of the alternative splicing of PAK1, a key MAPK signaling component. Mol. Cancer. 2017;16:175-017-0744-2. PubMed PMC

Vilas GL, et al. Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events. Proc. Natl. Acad. Sci. USA. 2006;103:6542–6547. doi: 10.1073/pnas.0600824103. PubMed DOI PMC

Kuzelova K, Grebenova D, Holoubek A, Roselova P, Obr A. Group I PAK inhibitor IPA-3 induces cell death and affects cell adhesivity to fibronectin in human hematopoietic cells. PLoS One. 2014;9:e92560. doi: 10.1371/journal.pone.0092560. PubMed DOI PMC

Licciulli S, et al. FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas. J. Biol. Chem. 2013;288:29105–29114. doi: 10.1074/jbc.M113.510933. PubMed DOI PMC

Roselova P, Obr A, Holoubek A, Grebenova D, Kuzelova K. Adhesion structures in leukemia cells and their regulation by Src family kinases. Cell. Adh Migr. 2018;12:286–298. doi: 10.1080/19336918.2017.1344796. PubMed DOI PMC

Sells MA, et al. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 1997;7:202–210. doi: 10.1016/S0960-9822(97)70091-5. PubMed DOI

Parrini MC, Camonis J, Matsuda M, de Gunzburg J. Dissecting activation of the PAK1 kinase at protrusions in living cells. J. Biol. Chem. 2009;284:24133–24143. doi: 10.1074/jbc.M109.015271. PubMed DOI PMC

Nicholas NS, et al. PAK4 suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells. Oncotarget. 2016;7:70881–70897. doi: 10.18632/oncotarget.12282. PubMed DOI PMC

Manser E, et al. Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes. Mol. Cell. Biol. 1997;17:1129–1143. doi: 10.1128/MCB.17.3.1129. PubMed DOI PMC

Frost JA, Khokhlatchev A, Stippec S, White MA, Cobb MH. Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation. J. Biol. Chem. 1998;273:28191–28198. doi: 10.1074/jbc.273.43.28191. PubMed DOI

Walter BN, et al. Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity. J. Biol. Chem. 1998;273:28733–28739. doi: 10.1074/jbc.273.44.28733. PubMed DOI

Lee N, et al. Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc. Natl. Acad. Sci. USA. 1997;94:13642–13647. doi: 10.1073/pnas.94.25.13642. PubMed DOI PMC

Mayhew MW, et al. Identification of phosphorylation sites in betaPIX and PAK1. J. Cell. Sci. 2007;120:3911–3918. doi: 10.1242/jcs.008177. PubMed DOI PMC

Sorrell, F. J., Kilian, L. M. & Elkins, J. M. Solution structures and biophysical analysis of full-length group A PAKs reveal they are monomeric and auto-inhibited in cis. Biochem. J (2019). PubMed PMC

Bright MD, Garner AP, Ridley AJ. PAK1 and PAK2 have different roles in HGF-induced morphological responses. Cell. Signal. 2009;21:1738–1747. doi: 10.1016/j.cellsig.2009.07.005. PubMed DOI

Kuzelova K, Grebenova D, Brodska B. Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes. J. Cell. Biochem. 2011;112:3334–3342. doi: 10.1002/jcb.23263. PubMed DOI

Deacon SW, et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem. Biol. 2008;15:322–331. doi: 10.1016/j.chembiol.2008.03.005. PubMed DOI PMC

Rennefahrt UE, et al. Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. J. Biol. Chem. 2007;282:15667–15678. doi: 10.1074/jbc.M700253200. PubMed DOI

Elsherif L, et al. Potential compensation among group I PAK members in hindlimb ischemia and wound healing. PLoS One. 2014;9:e112239. doi: 10.1371/journal.pone.0112239. PubMed DOI PMC

Lee JH, et al. HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases. Mol. Cell. 2013;49:668–679. doi: 10.1016/j.molcel.2012.12.004. PubMed DOI

Brodska B, Kracmarova M, Holoubek A, Kuzelova K. Localization of AML-related nucleophosmin mutant depends on its subtype and is highly affected by its interaction with wild-type NPM. PLoS One. 2017;12:e0175175. doi: 10.1371/journal.pone.0175175. PubMed DOI PMC

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