At least 99% of protein phosphorylation in eukaryotic cells occurs on Ser and Thr residues. The greater than 300 protein Ser/Thr kinases in the human genome are opposed by less than 30 protein Ser/Thr phosphatases, of which protein phosphatase 1 and protein phosphatase 2A (PP2A) 2 contribute the bulk of activity in most cell types. The notion of Ser/Thr phosphatases as promiscuous and constitutively active enzymes that simply provide the substrates for regulated kinase signaling has been challenged by the discovery of batteries of catalytic subunit-interacting proteins, which impart substrate specificity, subcellular localization, and responsiveness to phosphorylation (1, 2). In the case of PP2A, the predominant holoenzyme is formed by association of a core dimer of catalytic and scaffold subunits with one of a least 12 regulatory subunits in vertebrates. Since vertebrate A and C subunits are each encoded by two genes and since many regulatory subunits are diversified by alternative splicing, several dozen different PP2A heterotrimers are likely to exist in any given cell type. The three unrelated regulatory subunit gene families (B, BЈ, BЉ) are almost certain to utilize different mechanisms to control enzymatic activity and cellular localization of PP2A. The B-family (PR55) of regulatory subunits consists of predicted ␤-propellers with divergent N-terminal tails that act as subcellular targeting signals (3-5). In addition to interacting with phosphatase substrates (6 -12), BЈ-family (also referred to as B56, PR61) subunits are heavily phosphorylated, which may confer regulation by second messengers (13-16). Members of the BЉ-family of PP2A subunits (PR72/ 130, PR59, PR48) feature two calcium binding EF hands with presumed structural rather than regulatory functions (17). Striatin and SG2NA have been referred to as Bٞ regulatory subunits (18). It may be more appropriate to refer to them as PP2A dimer-associated proteins since their stability does not depend on association with the PP2A core enzyme (17, 19) and since they lack the A subunit binding consensus motif common to B, BЈ, and BЉ subunits (20).A growing body of evidence indicates that PP2A has complex inhibitory and stimulatory effects on hormone and growth factor signaling, in particular the extracellular-signal regulated (ERK)/mitogen-activated protein kinase (MAPK) cascade (21). PP2A substrates include G-protein-coupled receptors and receptor tyrosine kinases (22-24), receptorassociated proteins (25-28), and all three kinases of the ERK/MAPK cascade core module , MAPK/ERK kinase (MEK) (33)(34)(35) and ERK (36,37)).Here, we have begun to dissect the contribution of different PP2A holoenzymes to growth factor signaling in PC12 cells. The net effect of total PP2A silencing or inhibition was ERK and Akt hyperphosphorylation, most likely as a consequence of direct dephosphorylation of these kinases by PP2A. Cautioning against exclusive reliance on silencing approaches, protracted inhibition of PP2A by RNA interference (RNAi) resulted in a compensatory unco...

Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged B␣/␦ regulatory subunits by ϳ3-fold or knock-down of B␣ by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-B␣/␦ subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in B␣ knock-down cells but elevated in B␣-and B␦-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing B␣ or B␦. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes AB␣C and AB␦C act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A AB␣C and AB␦C holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1. PP2A3 is a major serine/threonine phosphatase implicated in the control of numerous cellular processes including metabolism, transcription and translation, ion transport, development, inflammation, cell growth, differentiation, and apoptosis (for reviews, see Refs. 1 and 2). Heterotrimeric PP2A holoenzymes are composed of a scaffolding/ structural subunit (A), a variable regulatory subunit (B), and a catalytic subunit (C). Thus far, four distinct regulatory subunit families have been identified: B or PR55 (2-4), BЈ or PR61 (5, 6), BЉ or PR72 (7-9), and Bٞ or PR93/PR110 (10). Although the regulatory subunit families share little amino acid sequence homology, isoforms within each family exhibit significant sequence homology. The variable subunit plays a critical role in the control of PP2A by regulating substrate selectivity and/or directing the localization of the enzyme within the cell (11-15). Recent studies have also revealed a role for PP2A regulatory subunits in cell growth and apoptosis (16 -18), assembly and function of cytoskeletal proteins (14,19,20), and various cell signal transduction pathways (for a review, see Ref. 2...

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links

Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3 (MAP2K), and a MAPK kinase kinase (MAP3K). Regulation of the MAP3K, presumably by phosphorylation, provides the impetus for activation of the three-kinase module. Once activated, the MAP3Ks activate MAP2Ks by phosphorylation of two residues within the activation loop. Phosphorylation of MAP2Ks activates these dual specificity kinases to phosphorylate MAPKs on a conserved threonine and tyrosine motif, TXY, also within the activation loop. Once phosphorylated, the MAPKs phosphorylate protein substrates and regulate cellular processes like growth, protein synthesis, gene expression, and nucleotide synthesis (2).Over the last decade, a large body of work has characterized events that occur downstream of the MAPKs. However, little is known regarding the regulatory mechanisms that modulate the MEKK proteins to ultimately regulate the MAPKs. For example, it is known that overexpression of MEKK3 activates the ERK (3, 4), JNK (3-5), p38 (5, 6), ERK5 (7), and NF-B pathways (8 -10). Typically, MEKK3-dependent regulation of these pathways is studied by using transfection studies, and activation of these pathways rarely requires an agonist. Therefore, it appears that some process intrinsic to MEKK3 is critical and sufficient for activation of these pathways.The activation loop of some protein kinase families, such as the arginine-aspartate family, is positioned between subdomains VII and VIII and is phosphorylated by other protein kinases or through autophosphorylation of the kinase itself (11). Phosphorylation within the activation loop of protein kinases results in conformational changes in the protein structure that (i) enhance substrate binding, (ii) correctly position amino acids involved in catalysis, and (iii) relieve steric hindrance within the catalytic domain. Regardless of how the activation loop is phosphorylated, regulation of catalytic activity frequently correlates with phosphorylation of the activation loop.Given that phosphorylation plays a critical role in regulating the MAPKs and the MAP2Ks, we investigated how phosphorylation of MEKK3 might affect its catalytic activity. Since phosphorylation sites within the activation loop of MEKK3 have not been reported, we systematically mutated serine and threonine residues within the activation loop to alanine and monitored MEKK3-dependent activities in HEK293 EBNA cells. Two key amino acids were identified at positions 526 and 530 using a luciferase-based reporter gene assay as well as assays that measure the ERK, JNK, and p38 MAP...