Table 2.

Phosphorylation sites of human PAK1 detected by mass spectrometry

Site	Detected without enrichment (+) or with IMAC	Homologs/other family members	Putative (roman) and known (italics) kinases
S2	+/IMAC	R, M, H/PAK2, PAK3
S21*	IMAC	R, M, H/PAK2, PAK3	AUTO/AKT
S57*	IMAC	R, M, H/PAK2, PAK3	AUTO
Y131*	+/IMAC	R, M, H/PAK2, PAK3	SRC
Y142	+/IMAC	R, M, H/PAK2, PAK3	PDGF-R
Y153	+/IMAC	R, M, H/PAK2, PAK3
S155/S156	+/IMAC	R, M, H/(S155) PAK 2 only
S174	+	R, M, H/PAK 3	Casein kinase 1
T185	+	R, M, H/PAK 2	ERK1/casein kinase 1
T212*	+/IMAC	R, M, H	ERK1, CDK5, CDC2
Y285	+	R, M, H/PAK 3	PY-SH2 motif
Y429	+/IMAC	R, M, H/PAK2, PAK3
Y464	IMAC	R, M, H/PAK2, PAK3	EGF-R/PY-SH2 motif

Serum-stimulated samples were treated with peroxovanadate and calyculin A to inhibit phosphatases. Numbered phosphorylation sites marked with (*) represents previously confirmed phosphorylation. Phosphorylation sites labeled ‘IMAC’ were only detected after sample enrichment by immobilized metal affinity chromatography. Sites detected without enrichment (C18) are denoted with a ‘+’. Homologous phosphorylation sites among species (R, rat; M, mouse; H, human) and PAK1 isoforms 1-3 were determined via sequence-comparison analysis using FASTA (http://fasta.bioch.virginia.edu/) (Pearson, 1990). Consensus phosphorylation motifs for putative kinases were analyzed using the Scansite scoring algorithm (http://scansite.mit.edu) (Obenauer et al., 2003). For additional data regarding PAK1 phosphorylation, see the Cell Migration Consortium web site: http://www.cellmigration.org