A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility

The dynamic assembly and disassembly of membrane cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.     

H2B (Ser10) Phosphorylation is Induced during Apoptosis and Meiosis in S. cerevisiae

The nucleosome, composed of an octamer of highly conserved histone proteins and associated DNA, is the fundamental unit of eukaryotic chromatin. How arrays of nucleosomes are folded into higher-order structures, and how the dynamics of such compaction are regulated, are questions that remain largely unanswered. Our recent studies demonstrated that phosphorylation of histone H2B is necessary to induce cell death that exhibits phenotypic hallmarks of apoptosis including DNA fragmentation and chromatin condensation in yeast (serine 10)1 and in mammalian cells (serine 14).2 In this article, we extend these findings by uncovering a role for H2B phosphorylation at serine 10 (Ser10) in another biological event that is associated with dramatic alterations in higher-order chromatin structure, meiosis. Our data show strong staining, indicative of H2B (Ser10) phosphorylation, during the pachytene stage of yeast meiotic prophase. These data broaden the use of this phosphorylation mark in chromatin remodeling that closely correlates with chromatin compaction. How phosphorylation marks are translated into meaningful downstream events during processes as diverse as apoptosis and meiosis remains a challenge for future studies.     

In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents

Insulin resistance is a key pathophysiologic feature of obesity and type 2 diabetes and is associated with other human diseases, including atherosclerosis, hypertension, hyperlipidemia, and polycystic ovarian disease. Yet, the specific cellular defects that cause insulin resistance are not precisely known. Insulin receptor substrate (IRS) proteins are important signaling molecules that mediate insulin action in insulin-sensitive cells. Recently, serine phosphorylation of IRS proteins has been implicated in attenuating insulin signaling and is thought to be a potential mechanism for insulin resistance. However, in vivo increased serine phosphorylation of IRS proteins in insulin-resistant animal models has not been reported before. In the present study, we have confirmed previous findings in both JCR:LA-cp and Zucker fatty rats, two genetically unrelated insulin-resistant rodent models, that an enhanced serine kinase activity in liver is associated with insulin resistance. The enhanced serine kinase specifically phosphorylates the conserved Ser(789) residue in IRS-1, which is in a sequence motif separate from the ones for MAPK, c-Jun N-terminal kinase, glycogen-synthase kinase 3 (GSK-3), Akt, phosphatidylinositol 3'-kinase, or casein kinase. It is similar to the phosphorylation motif for AMP-activated protein kinase, but the serine kinase in the insulin-resistant animals was shown not to be an AMP-activated protein kinase, suggesting a potential novel serine kinase. Using a specific antibody against Ser(P)(789) peptide of IRS-1, we then demonstrated for the first time a striking increase of Ser(789)-phosphorylated IRS-1 in livers of insulin-resistant rodent models, indicating enhanced serine kinase activity in vivo. Taken together, these data strongly suggest that unknown serine kinase activity and Ser(789) phosphorylation of IRS-1 may play an important role in attenuating insulin signaling in insulin-resistant animal models.     

Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2

Skp2 is the substrate binding subunit of the SCFSkp2 ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APCCdh1, which targets Skp2 for degradation in early G1 and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCFSkp2 ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.     

Characterisation of the phosphorylation of beta-catenin at the GSK-3 priming site Ser45

Activation of the canonical Wnt signalling pathway results in stabilisation and nuclear translocation of beta-catenin. In the absence of a Wnt signal, beta-catenin is phosphorylated at four conserved serine and threonine residues at the N-terminus of the protein, which results in beta-catenin ubiquitination and proteasome-dependent degradation. The phosphorylation of three of these residues, Thr41, Ser37, and Ser33, is mediated by glycogen synthase kinase-3 (GSK-3) in a sequential manner, beginning from the C-terminal Thr41. It has recently been shown that the GSK-3 dependent phosphorylation of beta-catenin requires prior priming through phosphorylation of Ser45. However, it is not known whether phosphorylation of Ser45 is carried out by GSK-3 itself or by an alternative kinase. In this study, the phosphorylation of beta-catenin at Ser45 was characterised using a phospho-specific antibody. GSK-3beta was found to be unable to phosphorylate beta-catenin at Ser45 in vitro and in intact cells. However, inhibition of GSK-3 in intact cells reduced Ser45 phosphorylation, suggesting that GSK-3 kinase activity is required for the phosphorylation event. In vitro, CK1, but not CK2, phosphorylates Ser45. Ser45 phosphorylation in intact cells is not mediated by CK1varepsilon, a known positive regulator of Wnt signalling, as overexpression of this kinase leads to decreased phosphorylation levels. In conclusion, phosphorylation of beta-catenin at the GSK-3 priming site Ser45 is not mediated by GSK-3 itself, but by an alternative kinase, indicating that beta-catenin is not an unprimed substrate for GSK-3 in vivo. Priming of GSK-3 dependent phosphorylation of beta-catenin by a different kinase could have important implications for the regulation of Wnt signalling.     

The dimeric versus monomeric status of 14-3-3zeta is controlled by phosphorylation of Ser58 at the dimer interface

The 14-3-3 proteins play a central role in the regulation of cell growth, cycling, and apoptosis by modulating the functional activities of key signaling proteins. Through binding to a phosphoserine motif, 14-3-3 alters target proteins activities by sequestering them, relocalizing them, conformationally altering their functional activity, or by promoting interaction with other proteins. These functions of 14-3-3 are facilitated by, if not dependent on, its dimeric structure. We now show that the dimeric status of 14-3-3 is regulated by site-specific serine phosphorylation. We found that a sphingosine-dependent kinase phosphorylates 14-3-3 in vitro and in vivo on a serine residue (Ser58) located within the dimer interface. Furthermore, by developing an antibody that specifically recognizes 14-3-3zeta phosphorylated on Ser58 and employing native-PAGE and cross-linking techniques, we found that 14-3-3 phosphorylated on Ser58 is monomeric both in vitro and in vivo. Phosphorylated 14-3-3 was detected solely as a monomer, indicating that phosphorylation of a single monomer within a dimer is sufficient to disrupt the dimeric structure. Significantly, phosphorylation-induced monomerization did not prevent 14-3-3 binding to a phosphopeptide target. We propose that this regulated monomerization of 14-3-3 controls its ability to modulate the activity of target proteins and thus may have significant implications for 14-3-3 function and the regulation of many cellular processes.     

Characterization of the expression of variant and standard CD44 in prostate cancer cells: Identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4–v10 variant CD44) in prostate cancer cells over‐expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT‐PCR, surface labeling with NHS–biotin, and immunoblotting. Expression of variant CD44 encompassing v4–v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT‐fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock‐down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent. J. Cell. Biochem. 108: 272–284, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor in intact cells

In a previous report we described Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor (IR) tyrosine kinase (TK) in vitro. The question remained whether the observed phosphorylation was exclusive for the in vitro activated receptor or a more general, mechanism of the activated receptor in situ. In this study, we determined the intrinsic activity of the IR to phosphorylate both serine residues in intact cells. For this purpose CHO-09 and NIH-3T3 derived cell-lines expressing the human IR were metabolically labelled with [(32)P]orthophosphate, followed by hormone stimulation of the receptor. The IR was isolated by immunoprecipitation and SDS-PAGE and subsequently analysed for serine phosphorylation by phosphopeptide mapping of HPLC-purified tryptic phosphopeptides. Activation of the IR in the intact cell appeared to result in phosphate incorporation into Ser(1275) and Ser(1309), providing strong evidence that both serine residues are phosphorylation sites of the activated receptor in intact cells.     

Phosphorylation of Grb10 by mitogen-activated protein kinase: identification of Ser150 and Ser476 of human Grb10zeta as major phosphorylation sites

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.     

The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily

PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCepsilon at residue Ser(368) controls an established PKCepsilon scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCepsilon and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.     

Negative regulation of the serine/threonine kinase B-Raf by Akt

B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.     

Differences in c-Jun N-terminal kinase recognition and phosphorylation of closely related stathmin-family members

The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif (⁴¹KKKDLSL⁴⁷) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP.     

Characterization of Ser338 phosphorylation for Raf-1 activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.     

Negative regulation of MAPKK by phosphorylation of a conserved serine residue equivalent to Ser212 of MEK1

The MAPKKs MEK1 and MEK2 are activated by phosphorylation, but little is known about how these enzymes are inactivated. Here, we show that MEK1 is phosphorylated in vivo at Ser(212), a residue conserved among all MAPKK family members. Mutation of Ser(212) to alanine enhanced the basal activity of MEK1, whereas the phosphomimetic aspartate mutation completely suppressed the activation of both wild-type MEK1 and the constitutively activated MEK1(S218D/S222D) mutant. Phosphorylation of Ser(212) did not interfere with activating phosphorylation of MEK1 at Ser(218)/Ser(222) or with binding to ERK2 substrate. Importantly, mimicking phosphorylation of the equivalent Ser(212) residue of the yeast MAPKKs Pbs2p and Ste7p similarly abrogated their biological function. Our findings suggest that Ser(212) phosphorylation represents an evolutionarily conserved mechanism involved in the negative regulation of MAPKKs.     

Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified. In a recent work, we revealed that DAPK2 is phosphorylated on Ser289 by the metabolic sensor AMPK, and that this phosphorylation enhances DAPK2 catalytic activity. Notably, Ser289 is conserved between DAPK1 and DAPK2, and was previously found to be phosphorylated in DAPK1 by RSK. Intriguingly, Ser289 phosphorylation was conversely reported to inhibit the pro-apoptotic activity of DAPK1 in cells. However, as the direct effect of this phosphorylation on DAPK1 catalytic activity was not tested, indirect effects were not excluded. Here, we compared Ser289 phosphorylation of the two kinases in the same cells and found that the intracellular signaling pathways that lead to Ser289 phosphorylation are mutually-exclusive and different for each kinase. In addition, we found that Ser289 phosphorylation in fact enhances DAPK1 catalytic activity, similar to the effect on DAPK2. Thus, Ser289 phosphorylation activates both DAPK1 and DAPK2, but in response to different intracellular signaling pathways.     

Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.