C/EBPalpha S248A mutation reduces granulocytic differentiation in human leukemic K562 cells

Phosphorylation of C/EBPα can either lead to granulocytic differentiation or a block in granulopoiesis. This dichotomy in effect is dependent on the upstream signaling pathway and the phosphorylation site in C/EBPα. Ras signaling induced phosphorylation of S248 residue of C/EBPα is known to enhance granulocytic differentiation. In this study, using β-estradiol inducible stable cell lines we show that the point mutation of phosphorylation site S248 in C/EBP disrupts the CD11b and GCSFr expression and subsequently reduces the differentiation of leukemic K562 cells. Based on our observations in the present study, we conclude that S248A mutation of C/EBPα leads to a reduction of granulocytic differentiation markers and a block in differentiation at the morphological level.     

Phosphorylation of CCAAT-enhancer binding protein by protein kinase C attenuates site-selective DNA binding.

Four DNA-recombinant proteins, corresponding to the DNA-binding domain of CCAAT/enhancer binding protein (C/EBP), were phosphorylated in vitro by protein kinase C (PKC). High-performance liquid chromatography-peptide mapping of 32P-labeled C/EBP indicated the presence of three major 32P-labeled peptides: S299 (P)RDK, AKKS277 (P)VDK, and GAAGLPGPGGS248 (P)LK. Phosphorylation of C/EBP by PKC or M-kinase resulted in an attenuation of binding to a 32P-labeled CCAAT oligodeoxynucleotide. Three other truncated forms of C/EBP, C/EBP87, C/EBP87S-C, and C/EBP60, were studied to define the sites of phosphorylation affecting DNA binding. Phosphorylation of the C/EBP87, containing sites Ser299 and Ser277, and C/EBP60, containing only site Ser299, by PKC also resulted in attenuation of DNA binding. In contrast, phosphorylation of C/EBP87S-C, which retained Ser277 but had a Cys in place of Ser299, had no effect on DNA binding. Ser299 could not be phosphorylated by PKC if the protein is already bound to specific DNA. Phosphorylation of intact C/EBP from liver nuclear extract by PKC or M-kinase occurred at Ser299 and Ser277 and at an additional site, as demonstrated by immunoprecipitation and peptide mapping.     

Functional regulation of thyroid hormone receptor variant TR alpha 2 by phosphorylation.

The thyroid hormone (T3) receptor (TR) variant TR alpha 2 is abundant in brain but does not bind T3 because of its unique C terminus. The only known function of TR alpha 2, inhibition of TR-dependent transactivation, involves competition for T3 response elements. Paradoxically, in vitro-translated TR alpha 2 bound poorly to these sites. We report here that dephosphorylation of TR alpha 2 restored its DNA binding. Mutation of C-terminal serine residues to alanine (TR alpha 2-SA) was equally effective. The C terminus of TR alpha 2 was phosphorylated in a human cell line, whereas that of TR alpha 2-SA was not. Conversely, TR alpha 2-SA was a much better inhibitor of T3 action than was wild-type TR alpha 2. The dominant negative activity of TR alpha 2-SA was less than stoichiometric with TR concentration, possibly because it was unable to heterodimerize with retinoid X receptor, which enhances the binding of other TRs. Purified casein kinase II as well as a reticulocyte casein kinase II-like activity phosphorylated TR alpha 2 on serines 474 and 475. Mutation of these two residues to alanine was sufficient to restore DNA binding. Thus, DNA binding by TR alpha 2 is regulated by phosphorylation at a site distant from the DNA-binding domain. The increased dominant negative activity of a nonphosphorylatable form of TR alpha 2 suggests that phosphorylation may provide a rapid, T3-independent mechanism for cell-specific modulation of the expression of T3-responsive genes.     

Regulation of Ras signaling specificity by protein kinase C

Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.     

Regulation of the Ras‐GTPase activating protein neurofibromin by C‐tail phosphorylation: implications for protein kinase C/Ras/extracellular signal‐regulated kinase 1/2 pathway signaling and neuronal differentiation

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase‐activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH‐SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Surprisingly, we observed that, among other PKC‐dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C‐tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA‐induced differentiation of SH‐SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C‐tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC‐dependent, sustained C‐tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.     

Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N‐terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine‐glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine‐alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation‐dependent regulation of translocation.     

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.