Recently, we have reported that the CacyBP/SIP protein binds ERK1/2 (Kilanczyk et al.. BBRC, 2009). In this work we show that CacyBP/SIP exhibits a phosphatase activity toward ERK1/2 kinases while its E217K mutant does not. The K-m and V-max values established for a standard phosphatase substrate, p-NPP, are 16.9 +/- 3.6 mM and 4.3 +/- 0.4 mu mol/min, respectively. The CacyBP/SIP phosphatase activity is decreased by okadaic acid (IC50 = 45 nM). Our experimental results are supported by a theoretical analysis which revealed important sequence similarities between CacyBP/SIP and the phosphatase-like proteins as well as certain MAP kinase phosphatases. (C) 2010 Elsevier Inc. All rights reserved.