alpha-4 was originally cloned from a λgt11 library using a monoclonal antibody made against a phosphoprotein that associated with the immunoglobulin alpha (Igα) protein in the BCR complex of immunoglobulin M cross-linked and phorbol myristate acetate-stimulated B cells (23
). alpha-4 is the homolog of the yeast protein called Tap42, which in yeast binds the type 2A protein phosphatases Pph21/22 (PP2A), Pph3 (PP4), and Sit4 (PP6) (12
). Tap42 functions in the TOR pathway in yeast to suppress phosphatase activity toward transcription factors Gln3 and Msn2 (5
). alpha-4 has also been shown to bind different type 2A protein phosphatases (PP2A, PP4, and PP6) in mammals (7
). We recently reported that alpha-4 exerts opposing kinetic effects on PP6 and PP2A (41
). Kong et al. demonstrated that conditional knockout of alpha-4 in thymocytes, as well as in other cell types, results in apoptosis (27
). Their conclusion was that alpha-4 acts as a nonredundant and dominant antiapoptotic factor in various tissues.
The p38 MAPK stress pathway has been implicated as a key regulator of apoptosis in cells stimulated with inflammatory cytokines (tumor necrosis factor alpha [TNF-α] or interleukin 1 [IL-1]) (1
). Programmed cell death, or apoptosis, can be engaged by either extrinsic or intrinsic pathways. The extrinsic pathway relies on activation of aspartyl proteases in response to inflammatory cytokines (TNF-α or IL-1) or other well-studied ligands, such as FasL. An initiator caspase in the inactive proform (e.g., procaspase 8) is bound via a death effector domain in an adapter protein associated with the C terminus of the receptor and is cleaved upon receptor activation, resulting in its activation (4
). This leads to activation of the downstream effector caspases 3 and 7 by proteolysis. The intrinsic pathway involves activation or inactivation of apoptogenic or proapoptogenic proteins involved in mitochondrially meditated apoptosis. These molecules consist of the Bcl-2 family members, functionally divided into two groups: the antiapoptotic Bcl-2 family members (Bcl-2 and Bcl-xL
) and the proapoptotic Bcl-2 family members (Bad, Bim, and Bax). Regulation of Bcl-2 or Bcl-xL
by p38 mitogen-activated protein kinase (MAPK) has been reported to cause their inactivation and to promote apoptosis (11
). Activation of Bad and BimEL
by p38 MAPK has also been shown (6
). Upon activation of proapoptotic proteins and inactivation of antiapoptotic proteins, cytochrome c
, apoptosis-inducing factor, and Smac/DIABLO are released from the mitochondria, resulting in activation of the Apaf complex (apoptosome) (4
). This promotes the activation of the initiator caspase 9, resulting in activation of the downstream effector caspases 3 and 7 and apoptosis (4
). Thus, assay of activated caspase 3/7 serves as a convenient readout of initiation of apoptosis by either pathway.
The MAPK family of proteins contains three kinases called ERK, p38 MAPK, and JNK. MAPKs are activated by upstream dual-specific serine/threonine and tyrosine kinases, referred to as MAPK/ERK kinases (MEKs), which are activated by MAPK kinase kinases (MKKKs) (8
). The ERK pathway is activated by growth factors that promote cellular growth and proliferation (8
). The p38 MAPK and JNK pathways are activated by cellular stress, such as inflammatory cytokines, hyperosmolality (sorbitol or NaCl), chemotherapeutics, chemicals (e.g., anisomycin), or UV irradiation (8
). These MAPK pathways are negatively regulated by dephosphorylation by multiple phosphatases (14
). Primarily, type 2A phosphatases catalyze the initial deactivation, whereas at later times following stimulation, dual-specificity MAPK phosphatases are induced and contribute to down-regulation of MAPK signals (14
). Evidence suggests that B regulatory subunits have a dominant role in targeting PP2A to ERK, especially B′ or B56 family members (29
). Regulatory-subunit specificity for JNK and p38 MAPK is not well understood.
To better understand the basis for alpha-4 action in apoptosis signaling, we sought to identify substrates of alpha-4-targeted phosphatases by depleting cells of alpha-4 using small interfering RNA (siRNA) and analyzing extracts with a multiplexed phosphorylation site screen offered by Kinexus, Inc. Knockdown of alpha-4 using siRNA selectively increased phosphorylation of p38 MAPK and c-Jun without changing ERK or JNK. We demonstrate that alpha-4 targets the p38 MAPK pathway by binding to the upstream kinase MEK3 and directing site-specific dephosphorylation of Thr193 in the activation loop. Overexpression of alpha-4 reduces cytokine activation of p38 MAPK and apoptosis and enhances cell growth in soft agar. The alpha-4 dominant-negative domain (DND) acts as an interfering protein to compete with endogenous alpha-4 to enhance cytokine activation of p38 MAPK and diminish cell growth in soft agar. The effects of alpha-4 depend on intact microtubules, as well as the alpha-4 binding partner Mid1, a protein mutated in the human disease Opitz syndrome. Thus, alpha-4 functions as a targeting and regulatory subunit for PP2A to oppose inflammatory-cytokine signaling to p38 MAPK by dephosphorylating and inactivating MEK3, thereby inhibiting apoptosis and anoikis.