Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization.

The interaction of tumor necrosis factor-alpha (TNFalpha) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42(mapk/erk2) phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207-425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFalpha, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42(mapk/erk2) and by specific inhibition of p42(mapk/erk2) activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42(mapk/erk2), and constitutively active MEK-1 followed by metabolic labeling with [(32)P]orthophosphate indicated that p42(mapk/erk2) phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42(mapk/erk2)-dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.     

Phosphorylation of the membrane proximal region of tumor necrosis factor receptor CD120a (p55) at ERK consensus sites

The interaction of tumor necrosis factor-alpha with its receptor CD120a (p55) initiates downstream signaling cascades that include the activation of the mitogen-activated protein kinase (MAPK), p42(mapk/erk2). The membrane proximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recent work, we showed that CD120a (p55) itself is a target of phosphorylation by p42(mapk/erk2), and after phosphorylation, the receptor is redistributed from the cell surface and Golgi complex to intracellular tubular structures associated with elements of the endoplasmic reticulum. The goal of this study was to define the specific amino acid residues that are phosphorylated. Deletional mutagenesis of the cytoplasmic domain of CD120a (p55) indicated that two sites located between residues 207-254 and 250-300 were phosphorylated predominantly on Thr and Ser residues, respectively. Site-directed mutagenesis of Ser and Thr residues contained within the extracellular signal-regulated kinase (ERK) consensus sequences indicated that the preferred residues were Thr-236 and Ser-270. Primary phosphorylation at these sites appeared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the preferred sites. Through the use of specific ligation of CD120a (p55) alone and mice deficient in CD120a (p55), CD120b (p75), or both receptors, CD120a (p55) was shown to be necessary and sufficient for the induction of kinase activity. These findings thus suggest that the phosphorylation of Thr-236 and Ser-270 within the membrane proximal region of CD120a (p55) are the preferred sites of phosphorylation by p42(mapk/erk2) and may set in motion phosphorylation at other sites.     

Hierarchical phosphorylation of the TNF-alpha receptor, TNF-R1, by p42Mapk/Erk at basic Pro-directed kinase sites

Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.     

Phosphorylation of the tumor necrosis factor receptor CD120a (p55) recruits Bcl-2 and protects against apoptosis

Ligation of the tumor necrosis factor alpha receptor CD120a initiates responses as diverse as apoptosis and the expression of NF-kappaB-dependent pro-survival genes. How these opposing responses are controlled remains poorly understood. Here we demonstrate that phosphorylation by p42(mapk/erk2) inhibits the apoptotic activity of CD120a while preserving its ability to activate NF-kappaB. Phosphorylated CD120a is re-localized from the Golgi complex to tubular structures of the endoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized the localization of CD120a to tubular structures and reversed the protection from apoptosis conferred by receptor phosphorylation. We propose that phosphorylation of CD120a represents a novel, Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42(mapk/erk2) may serve to inhibit the apoptotic activity of this death receptor while preserving NF-kappaB-dependent responses and may thus indirectly contribute to a failure to eliminate cells bearing oncogenes of the Ras-Raf-MEK-p42(mapk/erk2) pathway.     

Phosphorylation of 130- and 95-kDa substrates associated with tumor necrosis factor-alpha receptor CD120a (p55)

Cross-linking of CD120a (p55), a receptor for tumor necrosis factor alpha (TNFalpha), initiates downstream events, including the activation of protein Ser/Thr kinases. In this report, we have characterized two protein Ser/Thr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in their phosphorylation. pp130 and pp95 were detected by co-immunoprecipitation with CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays in the presence of [gamma-(32)P]ATP. The level of phosphorylation of pp130 and pp95 was rapidly and transiently increased in response to TNFalpha in [(32)P]orthophosphate-labeled macrophages, although the level of pp130 protein associated with CD120a (p55) remained unchanged as detected by [(35)S]methionine labeling. In contrast, pp130 and pp95 were efficiently phosphorylated in in vitro kinase assays of CD120a (p55) immunoprecipitates from unstimulated cells, and the level of phosphorylation was rapidly and transiently reduced in response to TNFalpha. Both pp130 and pp95 were sensitive to dephosphorylation with purified protein phosphatase 2A, and okadaic acid, a PP1/PP2A inhibitor, mimicked the ability of TNFalpha to stimulate the phosphorylation of pp130 and pp95 in intact (32)P-labeled macrophages. Collectively, these findings suggest that pp130 and pp95 are constitutively associated with CD120a (p55) and become inducibly phosphorylated in macrophages in response to TNFalpha. We propose that the underlying mechanism of their phosphorylation may involve the inactivation of a cytoplasmic pp130/pp95 Ser/Thr phosphatase.     

Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc gamma and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells

The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as FcgammaRII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in FcgammaRII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of FcgammaRII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fcgamma and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.     

Ordered phosphorylation of p42mapk by MAP kinase kinase.

Preparation of milligram amounts of [32P]p42mapk, phosphorylated at Tyr185 or diphosphorylated at Tyr185/Thr183, for use as specific protein phosphatase substrates is described. Tyr- but not Thr-phosphorylated p42mapk, accumulates when ATP is limiting. Furthermore, Tyr185-phosphorylated p42mapk exhibits an apparent 10-fold decrease in apparent Km (46.6 +/- 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (approximately 476 nM). We conclude that Tyr185 precedes Thr183 phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42mapk for MAP kinase kinase.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.     

Differential regulation of TNF-R1 signaling: lipid raft dependency of p42mapk/erk2 activation, but not NF-kappaB activation

The TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42(mapk/erk2) and NF-kappaB. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42(mapk/erk2) and NF-kappaB in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-alpha. Disruption of lipid rafts by cholesterol depletion prevented the TNF-alpha-dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42(mapk/erk2), while the activation of NF-kappaB was unaffected. In addition, phosphorylated p42(mapk/erk2), but not receptor interacting protein, I-kappaB kinase-gamma, or I-kappaBalpha was detected in raft-containing fractions following TNF-alpha stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.     

Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation

Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.     

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.     

Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.     

Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-alpha signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38(mapk) and the p46(jnk) and p54(jnk) c-jun NH(2)-terminal kinase isoforms, but not p42(mapk/erk2) or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42(mapk/erk2) and Akt by TNF-alpha was also inhibited in the presence of NaCl. Because the activation of p42(mapk/erk2) and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-alpha in the presence of NaCl compared with stimulation with TNF-alpha alone or NaCl alone. Furthermore, pharmacological inhibition of p42(mapk/erk2) and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.     

p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr(402) in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg(2+)/Mn(2+)-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.     

Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.     

Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(138) predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.     

Phosphorylation of phosphatase inhibitor-2 at centrosomes during mitosis

Inhibitor-2 (I-2) is a regulator of protein phosphatase type-1 (PP1), known to be phosphorylated in vitro by multiple kinases. In particular Thr72 is a Thr-Pro phosphorylation site conserved from yeast to human, but there is no evidence that this phosphorylation responds to any physiological signals. Here, we used electrophoretic mobility shift and immunoblotting with a site-specific phospho-Thr72 antibody to establish Thr72 phosphorylation in HeLa cells and show a 25-fold increase in phosphorylation during mitosis. Mass spectrometry demonstrated I-2 in actively growing HeLa cells was also phosphorylated at three other sites, Ser120, Ser121, and an additional Ser located between residues 70 and 90. In vitro kinase assays using recombinant I-2 as a substrate showed that the Thr72 kinase(s) was activated during mitosis, and sensitivity to kinase inhibitors indicated that the principal I-2 Thr72 kinase was not GSK3 but instead a member of the cyclin-dependent protein kinase family. Immunocytochemistry confirmed Thr72 phosphorylation of I-2 during mitosis, with peak intensity at prophase, and revealed subcellular concentration of the phospho-Thr72 I-2 at centrosomes. Together, the data show dynamic changes in I-2 phosphorylation during mitosis and localization of phosphorylated I-2 at centrosomes, suggesting involvement in mammalian cell division.     

Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G₂/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G₁ phase, whereas Ser387 was phosphorylated discontinuously in prophase and G₁ phase. Ser401 phosphorylation was enhanced in the G₁/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G₁-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.