Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38γ mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38γ, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38γ phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Rasdependent malignant growth in vitro and/or in mice by a mechanism depending on its p38γ-binding activity. Moreover, Ras increases both p38γ and PTPH1 protein expression and there is a coupling of increased p38γ and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38γ/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38␥ MAPK, and the activated c-Jun then recruits p38␥ as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38␥ that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38␥ interacting with c-Jun. p38␥ requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38␥ are required for the trans-activation of MMP9. The active p38␥/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38␥ and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response. MAPKs3 (including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38s) are critical signaling cascades that convert upstream signals into biological responses such as cell proliferation, invasion, and transformation (1). MAPKs are believed to do so by phosphorylating and activating a group of transcription factors, which through binding regulatory DNA elements lead to altered gene transcription. c-Jun is a major component of the AP-1 transcription factor downstream of MAPKs, whereas AP-1 is composed of homodimers of the Jun family or its heterodimers with another transcription factor such as c-Fos to bind the consensus DNA elements TGAg/cTCA (2). c-Jun is activated by JNK through phosphorylation at Ser-63, Ser-73, Thr-91, and Thr-93, and by ERK and p38 via increased gene expression. Activated c-Jun/ AP-1 leads to a cell type-specific biological response through integrated gene expression (1). However, the exact mechanism by which c-Jun converts a MAPK activity into a target gene expression remains mostly unknown.p38 MAPKs consist of four family members (␣, ␤, ␥, and ␦) in which p38␣ is ubiquitously present, whereas p38␥ is highly expressed in certain cancers (3). In addition to well established regulatory effects in cytokine signaling and stress response, substantial evidence suggests that the p38␣ pathway functions as a tumor suppressor (4 -8). p38␥, on the other hand, is a 43-kDa protein with an unique C-terminal motif, KETXL, that can dock with the PDZ (PSD-95/Dlg/ZO-1 homology) domain of other proteins (9, 10). In contrast to p38␣, our recent studies showed that p38␥ is induced by Ras and required for Ras transformation and invasion (11, 12), indicating its oncogenic activity. The underlying mechanisms for p38␥ involvement in Ras tumorigenesis, however, have not been established. In this report, we show that p38␥ acts both as an activator and a cofactor for c-Jun in trans-activating MMP9, a critical matrix metalloprote...

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Programmed cell death, or apoptosis, is inhibited by the antiapoptotic oncogene, Bcl-2, and is mediated by a cascade of aspartate-speci®c cysteine proteases, or caspases, related to interleukin 1-b converting enzyme. Depending on cell type, apoptosis can be induced by treatment with thapsigargin (TG); a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase. The role of caspases in mediating TG-induced apoptosis was investigated in the Bcl-2-negative human breast cancer cell line, MDA-MB-468. Apoptosis developed in MDA-MB-468 cells over a period of 24 ± 72 h following treatment with 100 nM TG, and was prevented by Bcl-2 overexpression. TG-induced apoptosis was associated with activation of caspase-3 and was inhibited by stable expression of the baculovirus p35 protein, an inhibitor of caspase activity. Also, TG-induced apoptosis was inhibited by treating cells with Z-VAD-fmk, a cellpermeable¯uoromethylketone inhibitor of caspases. These ®ndings indicate that TG-induced apoptosis of MDA-MB-468 breast cancer cells is subject to inhibition by Bcl-2 and is mediated by caspase activity. This model system should be useful for further investigation directed toward understanding the role of calcium in signaling apoptosis, and its relationship to Bcl-2 and the caspase proteolytic cascade.