Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Protein kinase D regulates RhoA activity via rhotekin phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

Protein kinase C regulates the phosphorylation and cellular localization of occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.     

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.     

Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.     

Identification of a new in vivo phosphorylation site in the cytoplasmic carboxyl terminus of EBV-LMP1 by tandem mass spectrometry

Latent membrane protein 1 (LMP1), an oncogenic protein encoded by Epstein-Barr virus (EBV), has been verified to be phosphorylated in vitro by protein casein kinase 2 (CK2). In this study, we characterized the phosphorylation of the carboxyl terminus of LMP1 fused with glutathione-S-transferase (GST-LMP1c) and the FLAG-epitope-tagged LMP1 (F-LMP1) proteins expressed in HEK293T cells. Using a combination of chemical modification and tandem mass spectrometry, we detected the phosphorylation of a tryptic peptide, 191-223 amino acids, in both GST-LMP1c catalysed by CK2 and F-LMP1-expressing cell lines. Serine residues at positions 211 and 215 were determined to be the substrates of CK2 in vitro. Most importantly, the S215 phosphorylation was also detected in F-LMP1-expressing human cell lines. The phosphorylation of S215, which is located in the carboxyl-terminus activation region 1 of LMP1, provides a new insight for investigating the role and modulation of the phosphorylation of LMP1.     

Expression and phosphorylation of the replication regulator protein geminin

It has been described that the replication regulator protein geminin is rapidly degraded at the end of mitosis and newly expressed at the beginning of the next S phase in the metazoan cell cycle. We have performed experiments to investigate the synthesis of geminin in cycling human HeLa cells. The levels of geminin-mRNA vary only modestly during the cell cycle with a 2-3-fold higher mRNA level at the G1/S phase transition, whereas newly synthesized geminin can only be detected in post-G1 phases. Surprisingly, geminin, once synthesized, does not remain stable, but is turned over during S phase with a half-life of 3-4h. We also show that geminin becomes phosphorylated as S phase proceeds and identify by MALDI mass spectrometry two specific major phosphorylation sites.     

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.     

Profilin oligomerization and its effect on poly (l-proline) binding and phosphorylation

Profilin is a cytoskeletal protein that interacts specifically with actin, phosphoinositides and poly (l-proline). Experimental results and in silico studies revealed that profilin exists as dimer and tetramer. Profilin oligomers possess weak affinity to poly (l-proline) due to unavailability of binding sites in dimers and tetramers. Phosphorylation studies indicate that profilin dimers are not phosphorylated while teramers are preferentially phosphorylated over monomers. In silico studies revealed that PKC phosphorylation site, S137 is buried in dimer while it is accessible in tetramer.     

蛋白质组中蛋白质磷酸化研究进展

随着后基因组时代的到来 ,对生命体器官、组织或细胞的全部蛋白质的表达、修饰及相互作用的研究已成为蛋白质组学的重要任务。蛋白质磷酸化是细胞内信号转导和酶调控最常见的机制之一 ,人类基因组约 2 %的基因编码 5 0 0种激酶和 10 0种磷酸酶。蛋白质磷酸化和去磷酸化作为原核和真核细胞表达调控的关键环节 ,了解其对功能的影响可以深入理解生命系统在分子水平的调控状况。目前蛋白质组磷酸化研究仍是功能基因组面临的重大课题 ,本文对此作一综述     

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Protein Kinase C (PKC) is a family of serine/threonine kinases whose function is influenced by phosphorylation. In particular, three conserved phosphorylation sites known as the activation-loop, the turn-motif and the hydrophobic-motif play important roles in controlling the catalytic activity, stability and intracellular localisation of the enzyme. Prevailing models of PKC phosphorylation suggest that phosphorylation of these sites occurs shortly following synthesis and that these modifications are required for the processing of newly-transcribed PKC to the mature (but still inactive) form; phosphorylation is therefore a priming event that enables catalytic activation in response to lipid second messengers. However, many studies have also demonstrated inducible phosphorylation of PKC isoforms at these sites following stimulation, highlighting that our understanding of PKC phosphorylation and its impact on enzymatic function is incomplete. Furthermore, inducible phosphorylation at these sites is often interpreted as catalytic activation, which could be misleading for some isoforms. Recent studies that include systems-wide phosphoproteomic profiling of cells has revealed a host of additional (and in many cases non-conserved) phosphorylation sites on PKC family members that influence their function. Many of these may in fact be more suitable than previously described sites as surrogate markers of catalytic activation. Here we discuss the role of phosphorylation in controlling PKC function and outline our current understanding of the mechanisms that regulate these phosphorylation sites.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches

The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1.     

Protein kinase C isozyme-specific phosphorylation of profilin

Profilin, a cytoskeletal protein, is emerging as an important link between signal transduction pathways and cytoskeletal dynamics. Profilin is phosphorylated on its C-terminal serine by protein kinase C (PKC). The protein kinase used for the in vitro phosphorylation studies reported earlier was a mixture of isozymes, and therefore, attempts were made to address the isozyme specificity on profilin phosphorylation under in vitro conditions. Profilin was subjected to phosphorylation by PKCalpha, PKCepsilon, and PKCzeta isozymes individually, and it was observed that profilin phosphorylation is cofactor-independent. PKCzeta phosphorylates profilin to a higher extent, but exhibits cofactor dependency with respect to phosphoinositides. The stoichiometry of phosphorylation was measured in the presence of these different isozymes, and a maximum stoichiometry of 0.8 (mole phosphate incorporated/mole profilin) was obtained in the presence of PKCzeta. Phosphorylation of profilin by PKCzeta was maximal in the presence of phosphatidylinositol4,5-bisphosphate (PI4,5-P2) when compared to the other phosphoinositides studied.     

C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins

MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells. It has been reported that the trafficking of MRP2 is modulated by PKC. Adjacent to the C-terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser(1542)). Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, beta2-syntrophin, and SAP-97). The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied. We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas beta2-syntrophin exhibited binding only to ABCA1. The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50. Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2.     

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.     

The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH(2)-terminal kinase pathway.

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH(2)-terminal kinase kinase (JNKK) and Jun NH(2)-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.