Abstract
Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2(-/-) MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2(-/-) MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2(-/-) MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G(2)/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2(-/-) mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.
Highlights
The kinetics of MAP kinase activation are strictly controlled principally by the mitogen-activated protein kinase phosphatases (MKPs),3 a family of at least 10 dual specific phosphatases (DUSPs) that function to terminate MAP kinase signaling within a defined subcellular location [7]
To confirm Mitogen-activated protein kinase phosphatase-2 (MKP-2) deletion, quiescent mouse embryonic fibroblasts from wild type or DUSP-4 deletion mice were stimulated with FCS over a period of 24 h (Fig. 1)
There was no increase in MKP-2 in stimulated mouse embryonic fibroblasts (MEFs) from the DUSP-4 KO mice (Fig. 1, A and B)
Summary
The kinetics of MAP kinase activation are strictly controlled principally by the mitogen-activated protein kinase phosphatases (MKPs),3 a family of at least 10 dual specific phosphatases (DUSPs) that function to terminate MAP kinase signaling within a defined subcellular location [7]. To ensure that MKP-2 protein expression was regulated in the normal manner in MKP-2ϩ/ϩ fibroblasts, we preincubated cells with different MAP kinase inhibitors prior to stimulation (Fig. 2). To confirm that the effect of DUSP-4 gene deletion had a universal effect upon proliferation, we assessed proliferation in two other cell types including adult CFs and mouse bone marrow macrophages (Fig. 7).
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