Phosphorylation Of Cytoplasmic Tail Research Articles

Canonical Wnt Pathway Is Activated in Myeloid Leukemia Stem Cells Via Hematopoietic Cell Kinase That Is Phosphorylated By an Autocrine TIM-3/Galectin-9 Signaling

(Introduction)Activation of β-catenin is critical for self-renewal of cancer stem cells (CSCs). A number of recurrent mutations related to the aberrant accumulation and activation of β-catenin have been identified in CSCs in various types of cancer, but such somatic mutations are barely found in leukemic stem cells (LSCs) in acute myeloid leukemia (AML). We have recently identified the autocrine loop consisted of LSC-specific surface molecule T-cell immunoglobulin mucin-3 (TIM-3) and its ligand galectin-9 (Gal-9) in human myeloid malignancies. When Gal-9 binds to TIM-3, TIM-3 signaling induces accumulation of β-catenin in nucleus (Kikushige et al, Cell Stem Cell, 2015). In this study, we sought to identify the molecular mechanism of aberrant β-catenin activation in LSCs of AML.(Results)To test whether TIM-3 signaling can activate the canonical Wnt pathway, we evaluated the activation of LDL receptor-related protein 6 (LRP6), a key component of canonical Wnt pathway. Immunoprecipitation assay by using anti-LRP6 antibody revealed that Gal-9 stimulation promoted the formation of the “LRP6-signalosome”, the protein complex consisted of Wnt components including LRP6, Axin and Dvl. Gal-9 stimulation also induced phosphorylation of LRP6at several sites including Thr1479 and subsequent β-catenin accumulation in primary TIM-3+ AML cells. On the other hand, this effect on LRP6 induced by Gal-9 stimulation was completely canceled in the presence of an anti-TIM-3 antibody (F38-2E2), which can block the TIM-3/Gal-9 interaction. Since the Wnt ligand is not involved in this assay system, the TIM-3/Gal-9 interaction should directly activate the canonical Wnt pathway, not through the previously identified Wnt signaling machinery.Gal-9 stimulation induced phosphorylation of cytoplasmic tail of TIM-3, which induces recruitment of several Src family kinases (SFKs). We found that Gal-9 ligation to TIM-3 activated several SFKs including hematopoietic cell kinase (HCK) by phosphorylation of the SH1 domain (Tyr410 in HCK) in TIM-3+ AML cells. Furthermore, an inhibitor specific for HCK, but not inhibitors effective for other SFKs, inhibited LRP6 phosphorylation induced by TIM-3/Gal-9 signaling, indicating that HCK should be a critical mediator for TIM-3 signaling in human LSCs.Next, we focused on p120-catenin because this molecule is known as not only a substrate for SFKs but also an indispensable molecule for the initial step of the LRP6-signalosome formation. We found that the phosphorylation of p120-catenin at Tyr228 was induced in response to Gal-9 stimulation. HCK inhibitors efficiently blocked the Tyr228 phosphorylation and subsequent activation of canonical β-catenin pathway. These results suggested that HCK activation induced by TIM-3/Gal-9 ligation could activate canonical β-catenin pathway through phosphorylation of p120-catenin.(Conclusion)In myeloid leukemia, TIM-3/Gal-9 signaling activates HCK, which induces p120-catenin phosphorylation, eventually acitavate the canonical β-catenin pathway, and these processes should be independent of Wnt ligands. Since TIM-3/Gal-9 autocrine loop signaling is constitutively active in AML, this novel machinery should contribute to the maintenance of AML LSCs through accumulation of β-catenin. Our study suggest that TIM-3 and Gal-9 as well as HCK should be important therapeutic targets in AML LCSs. DisclosuresNo relevant conflicts of interest to declare.

Tissue factor cytoplasmic tail regulates myeloid cell derived superoxide formation and TGF-beta 1 driven cardiac remodeling in myocardial infarction

Abstract Background In the setting of myocardial infarction (MI), patients with coronary no-reflow and/or delayed presentation after onset of symptoms (sub-acute MI) are inflicted by severe thrombo-inflammation and are marked by worse clinical outcome. However, it is unclear whether tissue factor (TF) contributes to outcome post MI solely by the regulatory functions of its cytoplasmic tail independently of its coagulation activity. Purpose We analyzed the role of the TF cytoplasmic domain in the recruitment of myeloid cells into the infarcted myocardium and the consequences on cardiac remodeling, scar formation, development of heart failure and survival post MI. Methods Twelve Patients enrolled in the MICAT (Mainzer Intracoronary Database, ClinicalTrials.gov Identifier: NCT02180178) study were examined. Patients with sub-acute MI and stable coronary artery disease were defined and monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Human heart samples acquired from the left ventricular wall of explanted hearts following cardiac transplantation or obtained during implantation of left ventricular assist device. Samples were investigated for downstream analysis of protein by western blots, RNA quantification and cryo-sectioning. MI was induced in 9 to 12 weeks old male C57BL/6J mice, mice specifically lacking the cytoplasmic tail (CT) of TF (TFΔCT mice) and TFfl/flLysMCre+/− mice by permanent ligation of the left anterior descending artery. Left ventricular function was assessed by High-Frequency Ultrasound System. Infiltration of immune cells into the infarcted myocardium was analyzed by performing flow cytometric analysis after enzymatic digestion of the myocardium. Superoxide levels were quantified by HPLC-based measurement of dihydroethidium derived oxidation product 2-hydroxy ethidium. Results Circulating monocytes in patients with sub-acute MI showed increased nitrosative stress as well as increased phosphorylation of TF CT along with TGF-β1 and NF-kB inflammatory activation, which was recapitulated in cardiac tissue of end-stage heart failure patients with chronic MI. MI results in phosphorylation of the CT of TF within myeloid cells. Using mice with conditional knockout of TF on myeloid cells or TFΔCT mice, we found that this regulatory intracellular domain of TF within myeloid cells is required for cardiac infiltration of inflammatory Ly6Chigh TF+ monocytes, Rac-1 GTPase and superoxide formation of gp91phox + myeloid cells in MI. TGF-β1 dependent SMAD2 activation and cardiac collagen deposition as late sequel of MI was reduced in TFΔCT mice, resulting in attenuated cardiac dysfunction and reduced mortality. Conclusion We conclude that, TF CT drives NADPH-oxidase derived superoxide formation, thrombo-inflammation and cardiac fibrosis. Therefore, it might serve as putative biomarker and risk predictor in MI Funding Acknowledgement Type of funding source: None

The C-terminal Phosphorylation Sites of eel Follicle-Stimulating Hormone Receptor are Important Role in the Signal Transduction.

The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phosphorylation sites exist in the C-terminal region of GPCRs. The experiments described herein represent attempts to determine the functions of the eel follicle-stimulating hormone receptor (eelFSHR). We constructed a mutant of eelFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 614 (eelFSHR-t614). The eelFSHR-t614 lacked all potential phosphorylation sites present in the C-terminal region of eelFSHR. In order to obtain the eelFSHR ligand, we produced recombinant follicle-stimulating hormone (rec-eelFSHβ/α) in the CHO-suspension cells. The expression level was 2-3 times higher than that of the transient expression of eelFSH in attached CHO-K1 cells. The molecular weight of the rec-eelFSHβ/α protein was identified to be approximately 34 kDa. The cells expressing eelFSHR-t614 showed an increase in agonist-induced cAMP responsiveness. The maximal cAMP responses of cells expressing eelFSHR-t614 were lower than those of cells expressing eelFSHR-wild type (eelFSHR-WT). The EC50 following C-terminal deletion in CHO-K1 cells was approximately 60.4% of that of eelFSHR-WT. The maximal response in eelFSHR-t614 cells was also drastically lower than that of eelFSHR-WT. We also found similar results in PathHunter Parental cells expressing β-arrestin. Thus, these data provide evidence that the truncation of the C-terminal cytoplasmic tail phosphorylation sites in the eelFSHR greatly decreased cAMP responsiveness and maximal response in both CHO-K1 cells and PathHunter Parental cells expressing β-arrestin.

Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering

The Hedgehog (Hh) signalling cascade is highly conserved and involved in development and disease throughout evolution. Nevertheless, in comparison with other pathways, our mechanistic understanding of Hh signal transduction is remarkably incomplete. In the absence of ligand, the Hh receptor Patched (Ptc) represses the key signal transducer Smoothened (Smo) through an unknown mechanism. Hh binding to Ptc alleviates this repression, causing Smo redistribution to the plasma membrane, phosphorylation and opening of the Smo cytoplasmic tail, and Smo oligomerisation. However, the order and interdependence of these events is as yet poorly understood. We have mathematically modelled and simulated Smo activation for two alternative modes of pathway activation, with Ptc primarily affecting either Smo localisation or phosphorylation. Visualising Smo activation through a novel, fluorescence-based reporter allowed us to test these competing models. Here, we show that Smo localisation to the plasma membrane is sufficient for phosphorylation of the cytoplasmic tail in the presence of Ptc. Using fluorescence cross-correlation spectroscopy (FCCS), we also demonstrate that inactivation of Ptc by Hh induces Smo clustering irrespective of Smo phosphorylation. Our observations therefore support a model of Hh signal transduction whereby Smo subcellular localisation and not phosphorylation is the primary target of Ptc function.

Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum

Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca(2+) which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca(2+) in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.

Phosphorylation of the Smo tail is controlled by membrane localization and is dispensable for clustering

The Hedgehog (Hh) signalling cascade is highly conserved and involved in development and disease throughout evolution. Nevertheless, in comparison with other pathways, our mechanistic understanding of Hh signal transduction is remarkably incomplete. In the absence of ligand, the Hh receptor Patched (Ptc) represses the key signal transducer Smoothened (Smo) through an unknown mechanism. Hh binding to Ptc alleviates this repression, causing Smo redistribution to the plasma membrane, phosphorylation and opening of the Smo cytoplasmic tail, and Smo oligomerisation. However, the order and interdependence of these events is as yet poorly understood. We have mathematically modelled and simulated Smo activation for two alternative modes of pathway activation, with Ptc primarily affecting either Smo localisation or phosphorylation. Visualising Smo activation through a novel, fluorescence-based reporter allowed us to test these competing models. Here, we show that Smo localisation to the plasma membrane is sufficient for phosphorylation of the cytoplasmic tail in the presence of Ptc. Using fluorescence cross-correlation spectroscopy (FCCS), we also demonstrate that inactivation of Ptc by Hh induces Smo clustering irrespective of Smo phosphorylation. Our observations therefore support a model of Hh signal transduction whereby Smo subcellular localisation and not phosphorylation is the primary target of Ptc function.

Termination of Protease-activated Receptor-1 Signaling by β-Arrestins Is Independent of Receptor Phosphorylation

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and beta-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, beta-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine(2A) receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, beta-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.

Association of Chemokine-mediated Block to HIV Entry with Coreceptor Internalization

Chemokines inhibit entry of HIV into CD4(+) T cells more effectively than into macrophages or transfected adherent cells. Here, we tested whether chemokine receptor internalization could account for cell type differences in the effectiveness of chemokines. Infection of CEM T cells expressing stably transduced wild-type CCR5 was much more readily inhibited by chemokine than were transduced HOS cells. This response correlated with the efficiency of CCR5 internalization. A mutated CCR5, termed M7-CCR5, in which the Ser/Thr phosphorylation sites in the cytoplasmic tail were changed to Ala, did not internalize in response to MIP-1alpha. M7-CCR5 was expressed at slightly higher levels than wild-type on stably transduced cell lines and was somewhat more potent as an HIV-1 coreceptor. The mutated receptor mobilized intracellular Ca(2+) in response to chemokine to a level 4-fold higher than did the wild type CCR5. Unexpectedly, the receptor was desensitized as efficiently as wild type, suggesting that desensitization does not require cytoplasmic tail phosphorylation. Entry of R5 HIV-1 reporter virus into cells stably expressing M7-CCR5 was largely resistant to blocking by MIP-1alpha. As much as 80% of entry inhibition was attributed to receptor internalization. Aminooxypentane (AOP)-MIP-1alpha was able to induce a low level of M7-CCR5 internalization in HOS and to weakly inhibit HIV-1 entry. Introduction of dominant negative dynamin into HOS cells reduced the ability of chemokine to inhibit infection. The inefficiency of internalization of chemokine receptors in some cell types could allow virus to replicate in vivo in the presence of endogenous chemokine. Last, M7-CCR5 is a useful tool for discriminating coreceptor internalization from binding site masking in the evaluation of small molecule inhibitors of HIV-1 entry.

Phosphorylation of a conserved integrin alpha 3 QPSXXE motif regulates signaling, motility, and cytoskeletal engagement.

Integrin alpha 3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a "QPSXXE" motif conserved in multiple alpha chains (alpha 3A, alpha 6A, alpha 7A), from multiple species. Phosphorylation of alpha 3A and alpha 6A did not appear to be directly mediated by protein kinase C (PKC) alpha, beta, gamma, delta, epsilon, zeta, or mu, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect alpha 3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) alpha 3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130(CAS) (in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) alpha 3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of alpha 3 and F-actin in CHO cells. Together, the results demonstrate clearly that alpha 3A phosphorylation is functionally relevant. In addition, the results strongly suggest that alpha 3 phosphorylation may regulate alpha 3 integrin interaction with the cytoskeleton.

Phosphorylation of the M2 Protein of Influenza A Virus Is Not Essential for Virus Viability

M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment.In vivoRa replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required forin vitroorin vivoreplication of influenza A virus.

Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization.

G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The alpha-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding alpha-factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize alpha-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.

Agonist-induced Sequestration, Recycling, and Resensitization of Platelet-activating Factor Receptor: ROLE OF CYTOPLASMIC TAIL PHOSPHORYLATION IN EACH PROCESS

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.

Immunolocalization of EGF receptor (EGFr) in intestinal epithelium: recognition of apoptotic cells

The EGF-like family of growth factors are known to be involved in the control of the intestinal epithelium. The intracellular events are mediated by the EGF receptor (EGFr), a transmembrane glycoprotein which is overexpressed in many malignancies and also in many radiosensitive cell types. The precise mode of action of the receptor in controlling proliferation and whether the factor is also involved in controlling apoptosis in this tissue is not clear. Using polyclonal antibodies raised against a cytoplasmic region of the receptor distant to the phosphorylation site and one raised against the peptide sequence DVVDADEYLIPQ, which is present in the cytoplasmic tail phosphorylation site of the EGFr, we have examined the immunostaining in normal and irradiated murine intestine. The former antibody labelled the basolateral membranes of the epithelial cells in the proliferative zones of both the small intestine and colon, in both control and irradiated tissue. The latter antibody however, strongly labelled the Goblet cells and the microvilli of the enterocyte apical membrane in control tissue. Following irradiation\ the apical labelling redistributed and was localized in the apical cytoplasm and in a paranuclear region. Furthermore, strong labelling was now seen in many of the apoptotic cells of the small intestinal epithelium. The greatly differing results with the two antibodies indicates that interpretation of such immunostaining must be viewed with caution and may relate to the availability of each particular epitope. These results also suggest that antibodies to DVVDADEYLIPQ may be a useful marker of apoptotic calls and could imply a correlation between high levels of epitope availability, the radiosensitive (frequently p53 expressing) cells of the crypt epithelium and the induction of apoptosis.

20 Domains of sAPP involved in various aspects of its biological activity

The relative utilization of alternative processing pathways for APP can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A, leads to a relative increase in utilization of the nonamyloidogenic, ‘α-secretase’ cleavage pathway for APP processing at the expense of other pathways. The molecular and cellular basis for this regulatory event is unknown. The possible mechanisms of regulated APP cleavage include (either singly or in combination): 1) substrate (ie APP) activation; 2) substrate redistribution; 3) enzyme (ie α-secretase) activation; or 4) enzyme redistribution. APP is a phosphoprotein; however, recent evidence from studies of the metabolism of mutant APP molecules suggests that changes in the APP cytoplasmic tail phosphorylation state may not be necessary for the phosphorylation-dependent activation of ‘α-secretase’ cleavage. Further, indirect immunofluorescent studies of the subcellular distribution of APP in the absence or presence of phorbol esters (PKC activators) fail to disclose obvious phorbol-induced redistribution of APP immunoreactivity. Taken together, current data suggest that major candidate phosphorylation-state sensitive targets relevant to the molecular basis of PKC-activated processing (or ‘regulated cleavage’) of APP include the APP ecto-domain as well as secretase enzymes and/or other components of the APP trafficking/processing apparatus. Progress in distinguishing among these possibilities is discussed.

23 Analysis of soluble, extracellular amyloid ß protein concentration in Alzheimer's disease

The relative utilization of alternative processing pathways for APP can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A, leads to a relative increase in utilization of the nonamyloidogenic, ‘α-secretase’ cleavage pathway for APP processing at the expense of other pathways. The molecular and cellular basis for this regulatory event is unknown. The possible mechanisms of regulated APP cleavage include (either singly or in combination): 1) substrate (ie APP) activation; 2) substrate redistribution; 3) enzyme (ie α-secretase) activation; or 4) enzyme redistribution. APP is a phosphoprotein; however, recent evidence from studies of the metabolism of mutant APP molecules suggests that changes in the APP cytoplasmic tail phosphorylation state may not be necessary for the phosphorylation-dependent activation of ‘α-secretase’ cleavage. Further, indirect immunofluorescent studies of the subcellular distribution of APP in the absence or presence of phorbol esters (PKC activators) fail to disclose obvious phorbol-induced redistribution of APP immunoreactivity. Taken together, current data suggest that major candidate phosphorylation-state sensitive targets relevant to the molecular basis of PKC-activated processing (or ‘regulated cleavage’) of APP include the APP ecto-domain as well as secretase enzymes and/or other components of the APP trafficking/processing apparatus. Progress in distinguishing among these possibilities is discussed.

Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization.

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.

Regulated cleavage of the Alzheimer amyloid precursor protein: Molecular and cellular basis

The relative utilization of alternative processing pathways for APP can be regulated by the activation state of certain protein phosphorylation signal transduction pathways. For example, activation of protein kinase C (PKC), or inactivation of protein phosphatases 1 and 2A, leads to a relative increase in utilization of the nonamyloidogenic, ‘α-secretase’ cleavage pathway for APP processing at the expense of other pathways. The molecular and cellular basis for this regulatory event is unknown. The possible mechanisms of regulated APP cleavage include (either singly or in combination): 1) substrate ( ie APP) activation; 2) substrate redistribution; 3) enzyme ( ie α-secretase) activation; or 4) enzyme redistribution. APP is a phosphoprotein; however, recent evidence from studies of the metabolism of mutant APP molecules suggests that changes in the APP cytoplasmic tail phosphorylation state may not be necessary for the phosphorylation-dependent activation of ‘α-secretase’ cleavage. Further, indirect immunofluorescent studies of the subcellular distribution of APP in the absence or presence of phorbol esters (PKC activators) fail to disclose obvious phorbol-induced redistribution of APP immunoreactivity. Taken together, current data suggest that major candidate phosphorylation-state sensitive targets relevant to the molecular basis of PKC-activated processing (or ‘regulated cleavage’) of APP include the APP ecto-domain as well as secretase enzymes and/or other components of the APP trafficking/processing apparatus. Progress in distinguishing among these possibilities is discussed.