Regulation of pigment organelle translocation. II. Participation of a cAMP-dependent protein kinase

In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore.     

Regulation of organelle membrane fusion by Pkc1p

Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers. The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p. While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated. We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C. Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation .     

Phosphorylation of the carotenoid droplet protein p57 by the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase and the effect of fluoride

We have previously shown that the dispersion and aggregation of carotenoid droplets in goldfish xanthophores are regulated, respectively, by phosphorylation and dephosphorylation of a carotenoid droplet protein p57. There is a basal level of p57 phosphorylation of p57 in unstimulated cells, which is greatly stimulated by adrenocorticotropic hormone (ACTH) or cyclic adenosine monophosphate (cAMP) acting via cAMP-dependent protein kinase. We have also observed that, in permeabilized xanthophores, pigment dispersion can be induced when cAMP is replaced by fluoride. Since p57 has multiple phosphorylation sites, there is the question of whether all p57 phosphorylation is by cAMP-dependent protein kinase or whether phosphorylation by cAMP-independent protein kinase coupled with inhibition of phosphatase activity by fluoride can replace cAMP-dependent protein kinase and that the ability of fluoride to replace cAMP for pigment dispersion in permeabilized cells is probably due to activation of adenylcyclase. We also show that ACTH causes an approximately threefold increase in the level of cAMP in these cells.     

Regulation of organelle acidity

Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674-687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 x 10(-6) cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance.     

Phosphorylation of components of the ER translocation site.

In many eukaryotic cells, protein secretion is regulated by extracellular signalling molecules giving rise to increased intracellular Ca2+ and activation of kinases and phosphatases. To test whether components involved in the first step of secretion, the translocation of proteins across the endoplasmic reticulum (ER) membrane, are regulated by Ca2+-dependent phosphorylation and dephosphorylation, we have investigated the effect of Ca2+ on kinases associated with the rough ER. Using purified rough microsomes from dog pancreas we found that Ca2+-dependent isoforms of protein kinase C (PKC) are associated with the rough ER and phosphorylate essential components of the protein translocation machinery. Phosphorylation of microsomal proteins by PKCs increased protein translocation efficiency in vitro. We also found that proteins of the translocation machinery became phosphorylated in intact cells. This suggests a further level of regulation of protein translocation across the ER membrane.     

Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.     

Protein phosphorylation and the two stages of pigment organelle dispersion in permeabilized xanthophores: organelle protein phosphorylation alone supports only the first stage

We reported previously that, in cultured goldfish xanthophores, dispersion of aggregated carotenoid droplets (CDs) requires the specific phosphorylation of the CD protein p57 by a cAMP-dependent protein kinase and the presence of cytosol. We report here that, in permeabilized cells, the addition of the catalytic subunit of cAMP-dependent protein kinase and ATP phosphorylates p57 and converts the CDs from an immobile to a mobile state (first stage of CD dispersion). However, the CDs are restricted to the vicinity of the original site of the CD aggregate and do not actually disperse (second stage of CD dispersion) unless cytosol is also added. We propose that this process may be related to aspects of secretory processes.     

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.     

Phosphorylation of the human cell proliferation-associated nucleolar protein p120

The human cell proliferation-associated nucleolar protein p120 was found in a variety of human cancer specimens but not in most normal resting cells. Polyclonal antibodies raised against bacterially expressed p120 were used to immunoprecipitate the p120 protein isolated from 32P-labeled HeLa cells. The p120 protein was phosphorylated at serine, threonine and tyrosine residues. A tryptic peptide map showed it contained three labeled peptides. One of these peptides comigrated with a p120 peptide phosphorylated in vitro by casein kinase II. This peptide was phosphorylated in vitro both at Ser-181 and Thr-185. This region is juxtaposed to the epitope site recognized by the anti-p120 monoclonal antibody.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Phosphorylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase C

CD20, a B cell integral membrane protein, regulates B cell activation and is differently phosphorylated in resting and activated cells. We have previously shown that CD20 phosphorylation is increased in activated cells and in phorbol ester-treated resting cells. Phosphorylation is also altered by agents which signal B cell proliferation, such as anti-IgM and a B cell growth factor. The present study was designed to address whether protein kinase C (PKC) or other kinases used CD20 as a substrate. When purified PKC was incubated with isolated CD20, both the 35- and 37-kDa CD20 proteins were phosphorylated in vitro. Intact resting B cells were next incubated with the protein kinase inhibitors H-7, H-8, and W-7. No change in basal CD20 phosphorylation was observed in the presence of W-7 and H-8, indicating that the protein cyclic nucleotide-dependent and calmodulin-dependent kinases were not actively phosphorylating CD20. Surprisingly, the PKC inhibitor H-7 increased CD20 phosphorylation at concentrations above 25-50 microM. To assess whether PKC either activated a phosphatase or inactivated a kinase affecting CD20 phosphorylation, tryptic phosphopeptide mapping of CD20 was performed. These studies revealed that addition of phorbol 12-myristate 13-acetate increased phosphorylation of some peptides differing from those which had increased phosphorylation following addition of H-7. Furthermore, signalling through surface immunoglobulin increased phosphorylation of CD20 peptides distinct from those hyperphosphorylated following addition of phorbol 12-myristate 13-acetate. These results demonstrate that 1) CD20 has multiple phosphorylation sites, as predicted from sequence data, and 2) whereas PKC can use CD20 as substrate, at least one other unidentified kinase phosphorylates CD20 in resting cells. Our data also predict that activation of B cells via the antigen receptor (surface IgM) may activate other protein kinases in addition to PKC.     

Phosphorylation of p36 in vitro by protein kinase C

The 36kDa subunit of protein I (p36) is a major substrate of several tyrosine protein kinases. Here we demonstrate that protein kinase C catalyzes the incorporation of 1.7 moles of phosphate per mole of protein I. Phosphorylation is absolutely dependent on the presence of both calcium and phospholipid, and is specific for serine and threonine residues. Phosphorylation of protein I by the c-AMP dependent protein kinase, phosphorylase kinase, casein kinase I, and casein kinase II was not observed. The in vivo significance of protein kinase C dependent phosphorylation of p36 is discussed.     

Phosphorylation of the yeast phospholipid synthesis regulatory protein Opi1p by protein kinase C

Opi1p is a negative regulator of expression of phospholipid-synthesizing enzymes in the yeast Saccharomyces cerevisiae. In this work, we examined the phosphorylation of Opi1p by protein kinase C. Using a purified maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase C activity was time- and dose-dependent, and dependent on the concentrations of Opi1p and ATP. Protein kinase C phosphorylated Opi1p on a serine residue. The Opi1p synthetic peptide GVLKQSCRQK, which contained a protein kinase C sequence motif at Ser(26), was a substrate for protein kinase C. Phosphorylation of a purified S26A mutant maltose-binding protein-Opi1p fusion protein by the kinase was reduced when compared with the wild-type protein. A major phosphopeptide present in purified wild-type Opi1p was absent from the purified S26A mutant protein. In vivo labeling experiments showed that the phosphorylation of Opi1p was physiologically relevant, and that the extent of phosphorylation of the S26A mutant protein was reduced by 50% when compared with the wild-type protein. The physiological consequence of the phosphorylation of Opi1p at Ser(26) was examined by measuring the effect of the S26A mutation on the expression of the phospholipid synthesis gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacI'Z reporter gene in opi1Delta mutant cells expressing the S26A mutant Opi1p was about 50% lower than that of cells expressing the wild-type Opi1p protein. These data supported the conclusion that phosphorylation of Opi1p at Ser(26) mediated the attenuation of the negative regulatory function of Opi1p on the expression of the INO1 gene.