Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220.
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The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme. (+info)
Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation.
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A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro. (+info)
Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity.
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Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment. (+info)
The protein phosphatase inhibitor cantharidin induces head and foot formation in buds of Cassiopea andromeda (Rhizostomae, Scyphozoa).
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The polyps of Cassiopea andromeda produce spindle shaped, freely swimming buds which do not develop a head (a mouth opening surrounded by tentacles) and a foot (a sticky plate at the opposite end) until settlement to a suited substrate. The buds, therewith, look very similar to the planula larvae produced in sexual reproduction. With respect to both, buds and planulae, several peptides and the phorbolester TPA have been found to induce the transformation into a polyp. Here it is shown that cantharidin, a serine/threonine protein phosphatase inhibitor, induces head and foot formation in buds very efficiently in a 30 min treatment, the shortest yet known efficient treatment. Some resultant polyps show malformations which indicate that a bud is ordinary polyp tissue in which preparatory steps of head and foot formation mutually block each other from proceeding. Various compounds related to the transfer of methyl groups have been shown to affect head and foot formation in larvae of the hydrozoon Hydractinia echinata. These compounds including methionine, homocysteine, trigonelline, nicotinic acid and cycloleucine are shown to also interfere with the initiation of the processes which finally lead to head and foot formation in buds of Cassiopea andromeda. (+info)
Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.
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Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. (+info)
Dephosphorylation of distinct sites on the 20 kDa myosin light chain by smooth muscle myosin phosphatase.
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The dephosphorylation of the myosin light chain kinase and protein kinase C sites on the 20 kDa myosin light chain by myosin phosphatase was investigated. The myosin phosphatase holoenzyme and catalytic subunit, dephosphorylated Ser-19, Thr-18 and Thr-9, but not Ser-1/Ser-2. The role of noncatalytic subunits in myosin phosphatase was to activate the phosphatase activity. For Ser-19 and Thr-18, this was due to a decrease in Km and an increase in k(cat) and for Thr-9 to a decrease in Km. Thus, the distinction between the various sites is a property of the catalytic subunit. (+info)
Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.
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A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme. (+info)
Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex.
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Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes. (+info)