Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.     

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.     

Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori

From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family. Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins. The other 4 proteins show low sequence similarity to any of the known Rab proteins. However, all of them contain the region conserved in rab protein. Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated. The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column. The protein bound [(3)H]-GDP with association constant of 1.02 x 10(11) M(-1). Further, the protein was phosphorylated by protein kinase. This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation.     

Phosphorylation by Protein Kinase C of the Muscarinic Acetylcholine Receptor

Muscarinic acetylcholine receptors purified from porcine cerebrum were phosphorylated by protein kinase C purified from the same tissue. More than 1 mol of phosphate was incorporated per mole of receptor, with both serine and threonine residues being phosphorylated. Neither the degree nor the rate of the phosphorylation was affected by the presence or absence of acetylcholine. GTP-sensitive high-affinity binding with acetylcholine was observed for muscarinic receptors reconstituted with GTP-binding proteins (Gi or Go), irrespective of whether muscarinic receptors or the GTP-binding proteins had been phosphorylated by protein kinase C or not. This indicates that the interaction between purified muscarinic receptors and purified GTP-binding proteins in vitro is not affected by their phosphorylation.     

Phylogenetically conserved CK-II phosphorylation site of the murine homeodomain protein Hoxb-6.

In an effort to characterize the signal transduction mechanisms that operate to regulate homeodomain protein function, we have analyzed the phosphorylation state of two homeodomain proteins, Hoxb-6 and Hoxc-8, in vitro and in vivo. The baculovirus expression system was employed to demonstrate that Hoxb-6 is phosphorylated in Sf9 cells while Hoxc-8 is not. Using two-dimensional tryptic phosphopeptide mapping and purified protein kinases, we demonstrate that Hoxb-6 is phosphorylated in vitro by casein kinase II and cAMP-dependent protein kinase. The casein kinase II phosphorylation site was mapped to serine-214. Two-dimensional tryptic phosphopeptide mapping of immunoprecipitated Hoxb-6 from mouse embryonic spinal cords demonstrates that the same peptide phosphorylated in vitro and in Sf9 cells by casein kinase II is also phosphorylated in vivo. The conservation of this site in several homeodomain proteins from various species is discussed.     

Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase

The substrate specificity of protein kinase C was studied and compared with that of cyclic AMP-dependent protein kinase (protein kinase A) by using bovine brain myelin basic protein as a model substrate. This basic protein was phosphorylated at multiple sites by both of these protein kinases. In this analysis, the basic protein was thoroughly phosphorylated in vitro with [gamma-32P]ATP and each protein kinase, and then digested with trypsin. The resulting radioactive phosphopeptides were isolated by gel filtration followed by high performance liquid chromatography on a reverse-phase column. Subsequent amino acid analysis and/or sequential Edman degradation of the purified phosphopeptides, together with the known primary sequence of this protein, revealed that Ser-46 and Ser-151 were specifically phosphorylated by protein kinase C, whereas Thr-34 and Ser-115 were phosphorylated preferentially by protein kinase A. Both kinases reacted with Ser-8, Ser-11, Ser-55, Ser-110, Ser-132, and Ser-161 at various reaction velocities. Contrary to protein kinase A, protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine. The seryl residues that are phosphorylated commonly by these two protein kinases have basic amino acids at both the amino- and carboxyl-terminal sides. These results provide some clues to understanding the rationale that these kinases may show different but sometimes similar functions depending on the structure of target phosphate acceptor proteins.     

Phosphorylation of rhodopsin by protein kinase C in vitro

Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.     

A GAPDH mutant defective in Src-dependent tyrosine phosphorylation impedes Rab2-mediated events

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2-mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2-dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane-binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose-dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus-G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C iota/lambda to GAPDH Y41F, which reduces beta-coat protein association with the VTC and subsequent formation of Rab2-mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.     

Putative regulatory mechanism of prothoracicotropic hormone (PTTH) secretion in the American cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis> as inferred from co-localization of Rab8, PTTH,and protein kinase C in neurosecretory cells

Small GTPases of the Rab family act as essential regulators of vesicle transport pathways, including the exocytosis of neurohormones. These processes are not well-understood in insects. To address the physiological function of Rab proteins and their phosphorylation in insect neurosecretion, Rab8-like, prothoracicotropic hormone (PTTH)-like, and protein kinase C (PKC)-like immunohistochemical reactivities (-ir) were investigated in the brain of the American cockroach, Periplaneta americana. All the antibodies tested reacted with neurons in the pars intercerebralis, corpora cardiaca, and nervi corporis allati I. Double-labeling experiments demonstrated that all PTTH-ir were colocalized with Rab8-ir and PKC-ir in the pars intercerebralis, although exclusive reactivity was present to antisera against Rab8 or PKC. These findings support the notion that Rab8-like antigen is phosphorylated by PKC, and that this phosphorylation is involved in the axonal transport and secretion of PTTH in this species. This work was supported by a Grant-in-Aid for Scientific Research (B) 1838043, (C) 20580053, and Global Center of Education and Research Program (19GCOE01) from the Japan Society for the Promotion of Science.     

Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C.

A Mr-80,000 acidic phosphoprotein ('80K protein') is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

Involvement of protein kinase C in the phosphorylation of rhodopsin

The possibility that protein kinase C is involved in phototransduction by phosphorylating rhodopsin was explored in situ and in vitro. Pretreatment of intact retinas with phorbol myristate acetate markedly increased the light-dependent phosphorylation of rhodopsin, with the greatest effects observed at lower light levels. Phorbol myristate acetate treatment did not affect rhodopsin phosphorylation in retinas not exposed to light, suggesting that protein kinase C modulates the phosphorylation state of rhodopsin in a light-dependent manner. Limited proteolysis of rhodopsin phosphorylated in situ indicates that protein kinase C modifies rhodopsin on a domain distinct from that recognized by rhodopsin kinase. In vitro, protein kinase C purified from bovine retinas phosphorylated unbleached and bleached rhodopsin. Our results are consistent with protein kinase C phosphorylating unbleached rhodopsin in response to low light, suggesting that protein kinase C plays a role in light adaptation.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.