The class I p21-activated kinases (Pak1-3) regulate many essential biological processes including cytoskeletal rearrangement cell cycle progression apoptosis and cellular transformation. A characteristic that distinguishes the atypical MAPKs from the classical MAPK isoforms is the lack of the characteristic “T(16). However a cell fractionation approach to identify the kinases responsible for Erk3 Ser189 phosphorylation suggested that the cellular Ser189 kinase did not co-fractionate with Erk1/2 kinase activity excluding Mek1/2 from consideration as cellular Erk3 kinases (17). Similar SAHA approaches excluded protein kinase C (PKC) as the cellular Ser189 kinase despite the ability of PKC to phosphorylate Erk3 (17). The initial observation of Erk3 Ser189 phosphorylation occurred more than a decade ago (17) but the kinase(s) responsible for this phosphorylation remain unidentified. Using high density protein microarrays we identified the Erk3 atypical MAPK as a candidate Pak2 substrate. Pak2 kinase assays using recombinant full-length Erk3 protein in solution confirmed the protein microarray results and suggested that Erk3 is a direct p21-activated kinase substrate and suggesting a role for class I p21-activated kinases in regulating the subcellular localization and activity of the atypical MAPK Erk3. EXPERIMENTAL PROCEDURES Human Protein Microarrays Kinase substrate identification (KSI) ProtoArrays (version 4.0 arrays containing 8 274 full-length GST fusion proteins provided by Invitrogen) were processed as recommended by the manufacturer with the following modification; the supplied kinase buffer was replaced with 1× phosphobuffer (19). Recombinant His6-Pak2 expressed from was used as the exogenous kinase. This source of Pak2 is known to be constitutively active3 obviating the need for inclusion of Rac/Cdc42-GTP. “ATP only” and “ATP + Pak2” slides were identically processed in parallel. Radiographic images of the slides were obtained using a Fuji phosphorimager and spots were identified using GenePix Pro (Molecular Devices). Data analysis was conducted in ProtoArray Prospector version 2.0 software as recommended SAHA (Invitrogen). For overlay images spots were pseudocolored in Adobe Photoshop. Plasmids and Plasmid Construction The human Erk3 vector was kindly provided by Dr. Sylvain Meloche University of Montreal. The Erk3 sequence was cloned into the N-terminal His6 baculovirus transfer vector pFBHTB and used to make high titer baculovirus stocks (P4) as recommended (Invitrogen). For mammalian expression vectors a human full-length Erk3 ultimate ORF Gateway entry clone was purchased from DHCR24 Invitrogen and shifted to the N-terminal GFP (pcDNA6.2-EmGFP-N) as well as the N-terminal His6 (pDest26) Gateway destination vectors using the LR Clonase II enzyme mix as recommended (Invitrogen). Site-directed stage mutants of Erk3 (S189A S189D) had been made out of the Erk3 best ORF plasmid as template SAHA and QuickChange II package (Stratagene) with the next mutagenic oligonucleotides: SA1 (5′-cattattcccataagggtcatcttgctgaaggattggttactaaat-3′); SA2 (5′-atttagtaaccaatccttcagcaagatgacccttatgggaataatg-3′); SD1 (5′-ctcattattcccataagggtcatcttgatgaaggattggttactaaatgg-3′); and SD2 (5′-ccatttagtaaccaatccttcatcaagatgacccttatgggaataatgag-3′). Plasmids encoding just the required S189A or S189D mutations had been determined by DNA sequencing and had been then shifted to pcDNA6.2-EmGFP-N and pDest26 vectors as described over. GFP-PID SAHA (matching to proteins 83-149 of individual Pak1) and GFP-PIDL107F constructs had been amplified by PCR using individual Pak1 and Pak1L107F appearance constructs as template and eventually cloned into pEGFP-C1 (Clontech). mRFP-PID constructs had been built by Pfusion (New Britain Biolabs) PCR using the above mentioned GFP-PID vectors as web templates and the next oligonucleotides 5 and 5′-gcgaattcctatttatctgtaaagctcatgtattt-3′. The PCR items had been digested with BamHI and EcoRI limitation enzymes purified from 1× TBE/agarose gels and ligated into computers2 + mRFP-N1 (something special from Dr. Chris Thorpe Kansas Condition College or university) similarly digested. Applicant clones had been identified by limitation analysis accompanied by DNA series confirmation. Purification and Dephosphorylation of Recombinant Erk3 High titer P4 stocks were used to infect suspension cultures of Sf21 cells managed in SFM-900 III media (Invitrogen). 48 h post-infection the cultures were pelleted by centrifugation and the supernatant was discarded and the pellet.