Regulatory Phosphorylation Sites Research Articles

Identification of a regulatory phosphorylation site in a cell cycle and swelling regulated ClC Cl‐ channel

clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans voltage‐gated ClC Cl‐ channel. CLH‐3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC‐7α and GLC‐7β. GCK‐3 is a Ste20 kinase and homolog of mammalian PASK/SPAK, which regulates cation‐coupled Cl‐ cotransporters. GCK‐3 binds to a 101 amino acid C‐terminal splice insert unique to CLH‐3b. When co‐expressed with CLH‐3b, GCK‐3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Knockdown of GCK‐3 in worm oocytes constitutively activates CLH‐3b. Using mass spectrometry, we identified a serine residue (S747) located within the 101 amino acid splice insert that is phosphorylated in the presence of GCK‐3. S747 and surrounding amino acids conform to the Ste20 kinase phosphorylation motif. Mutation of S747 to alanine prevents GCK‐3 mediated channel inhibition and alterations of channel voltage‐dependent properties. We conclude that phosphorylation of S747 is critical for regulation of CLH‐3b activity. S747 may be phosphorylated directly by GCK‐3 or other downstream kinases.Supported by NIH R01 grant DK51610

Structurally Distinct Phosphatases CD45 and CD148 Both Regulate B Cell and Macrophage Immunoreceptor Signaling

The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K–PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K–PKB pathways, the importance of both lipids must be considered.

Protein kinase C delta is phosphorylated on five novel Ser/Thr sites following inducible overexpression in human colorectal cancer cells

Phosphorylation plays an important role in regulation of protein kinase C delta (PKCdelta). To date, three Ser/Thr residues (Thr 505, Ser 643, and Ser 662) and nine tyrosine residues (Tyr 52, Tyr 64, Tyr 155, Tyr 187, Tyr 311, Tyr 332, Tyr 512, Tyr 523, and Tyr 565) have been defined as regulatory phosphorylation sites for this protein (rat PKCdelta numbering). We combined doxycycline-regulated inducible gene expression technology with a hypothesis-driven mass spectrometry approach to study PKCdelta phosphorylation pattern in colorectal cancer cells. We report identification of five novel Ser/Thr phosphorylation sites: Thr 50, Thr 141, Ser 304, Thr 451, and Ser 506 (human PKCdelta numbering) following overexpression of PKCdelta in HCT116 human colon carcinoma cells grown in standard tissue culture conditions. Identification of potential novel phosphorylation sites will affect further functional studies of this protein, and may introduce additional complexity to PKCdelta signaling.

RCC1 isoforms differ in their affinity for chromatin, molecular interactions and regulation by phosphorylation

RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Generation of Ran-GTP by RCC1 on chromatin provides a spatial signal that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. We show that RCC1 is expressed in human cells as at least three isoforms, named RCC1alpha, RCC1beta and RCC1gamma, which are expressed at different levels in specific tissues. The beta and gamma isoforms contain short inserts in their N-terminal regions (NTRs) that are not present in RCC1alpha. This region mediates interaction with chromatin, binds importin alpha3 and/or importin beta, and contains regulatory phosphorylation sites. RCC1gamma is predominantly localised to the nucleus and mitotic chromosomes like RCC1alpha. However, compared to RCC1alpha, RCC1gamma has a greatly reduced interaction with an importin alpha3-beta and a stronger interaction with chromatin that is mediated by the extended NTR. RCC1gamma is also the isoform that is most highly phosphorylated at serine 11 in mitosis. Unlike RCC1alpha, RCC1gamma supports cell proliferation in tsBN2 cells more efficiently when serine 11 is mutated to non-phosphorylatable alanine. Phosphorylation of RCC1gamma therefore specifically controls its function during mitosis. These results show that human RCC1 isoforms have distinct chromatin binding properties, different molecular interactions, and are selectively regulated by phosphorylation, as determined by their different NTRs.

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

Major Cdk5‐dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis

Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.

ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

The regulation of smooth muscle contractility by zipper-interacting protein kinaseThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.

C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

PKCδ-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCδ on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCδ-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCδ catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

Akt Signaling and Growth of the Heart

Physiological and pathological stimuli produce clinically and molecularly distinct forms of cardiac growth. Physiological cardiac growth is a feature of normal postnatal development in which an increase in cardiac muscle cell diameter is observed as infants mature to adults.1 This nonpathological heart growth, sometimes referred to as physiological hypertrophy,2 is similar to the growth observed in the hearts of trained athletes in whom the adaptation to increased workload leads to increased vascularization of the myocardium and more forceful ejection.1,3 In contrast, pathological hypertrophy occurs in patients with hypertension or valvular heart disease. A number of molecular distinctions can be made between physiological and pathological cardiac hypertrophy. For example, pathological cardiac hypertrophy is associated with interstitial fibrosis, activation of a fetal gene program, and myocyte apoptosis, whereas physiological cardiac growth does not display these features. Most studies have focused on elucidating mechanisms of pathological heart growth, whereas the molecular regulation of physiological cardiac growth is less understood. In this issue of Circulation , an article by DeBosch et al4 sheds light on the role of Akt1 signaling in the promotion of physiological growth and inhibition of pathological hypertrophy. Article p 2097 The Akt (also referred to as protein kinase B) family of serine/threonine protein kinases is highly conserved in evolution.5 Akt1 and Akt2 share extensive sequence homology at the amino acid level, whereas Akt3 is slightly more divergent in structure and is expressed as a splice variant that lacks a regulatory phosphorylation site.6 Akt protein kinases are stimulated by a number of receptor tyrosine kinases, and this is mediated by the action of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates inositol lipids that activate Akt directly by binding to its pleckstrin homology domain and promoting its translocation to the membrane …

P190 RhoGAP functions in tissue morphogenesis

The p190 family of Rho GTPase activating proteins (GAPs) includes two closely related proteins, p190A and p190B, that are potent inhibitors of Rho GTPase activity. They can each be regulated in response to the engagement of cell surface adhesion molecules, such as integrins, as well as by activated growth factor receptors, and they play essential roles in mouse embryogenesis. However, they appear to mediate distinct signaling pathways by virtue of non-overlapping sites of regulatory phosphorylation. For p190A, src kinases are important regulators of activity and p190A is the principal substrate of src kinase-mediated phosphorylation in the brain. In addition, growth factor receptors, such as the PDGF receptor, can directly phosphorylate p190A on one of its so-called FF domains. As a consequence, p190A dissociates from the transcription factor, TFII-I, which becomes free to translocate to the nucleus to activate gene transcription. p190B is regulated through distinct mechanisms. p190B can be directly phosphorylated on a single identified tyrosine residue by the activated insulin and IGF-1 receptors. This phosphorylation causes p190B to translocate to the lipid rafts of the plasma membrane, where it can potentially facilitate the inactivation of the Rho GTPase, which also becomes enriched within the lipid rafts. Thus, upon insulin/IGF-1 signaling, phosphorylation of p190B can promote Rho inactivation. Mechanisms by which p190B may function to link IGF-1 and integrin-mediated signals that influence Rho-dependent actin reorganization will be presented.

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors <2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with (15)N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

CAMP signal transduction induces eNOS activation by promoting PKB phosphorylation

The objective of this study was to determine whether activation of protein kinase B (PKB) is involved in the production of nitric oxide (NO) induced by cAMP signal transduction. Mongrel dogs were used for this study. Coronary microvessels were isolated from the left ventricular free wall of these dog hearts. Forskolin (an activator of adenylyl cyclase that increases intracellular cAMP level) and 8-bromo-cAMP (a membrane-permeable cAMP analog) were used to stimulate NO release and activation of PKB and endothelial NO synthase (eNOS) in these blood vessels. We found that forskolin and 8-bromo-cAMP increased NO release (quantified by using the Griess reaction) from coronary microvessels by 80 +/- 6 and 78 +/- 11 pmol/mg (mean +/- SE), respectively (P < 0.05 vs. control). Western blot analysis showed that forskolin elicited a significant increase in eNOS phosphorylation (59 +/- 11%) at serine-1177 (a positively regulatory phosphorylation site for eNOS) and a significant increase in dephosphorylation (28 +/- 6%) at threonine-495 (a negatively regulatory phosphorylation site of eNOS) (P < 0.05 vs. control). Interestingly, forskolin also increased the phosphorylation of PKB at serine-473 (by 49 +/- 17%) and threonine-308 (by 53 +/- 17%), respectively (P < 0.05 vs. control; phosphorylation of both sites is required for a full activation of PKB). N(omega)-nitro-l-arginine methyl ester (an NOS inhibitor) blocked NO formation, Rp diastereomer of cAMP (a PKA inhibitor), and LY-294002 [a PI3-kinase (an activator of PKB) inhibitor] prevented the production of NO, phosphorylation of PKB, and eNOS induced by forskolin. Our data clearly show an involvement of PKB activation in cAMP signal-induced NO production. We are reporting for the first time that cAMP signal transduction stimulates eNOS activation through a PKB-mediated mechanism.

Beta-Catenin mutation and its nuclear localization are confirmed to be frequent causes of Wnt signaling pathway activation in pilomatricomas

beta-Catenin has been shown to play an important role in the formation of hair follicle-related tumors, including pilomatricomas. Several investigators have shown that beta-catenin gene mutation is observed in pilomatricomas. However, the relationship between the pattern of beta-catenin localization in the cell and beta-catenin gene mutation is still controversial. This work was performed to determine the frequency of beta-catenin nuclear localization in pilomatricoma, the relationship between the pattern of beta-catenin localization and beta-catenin mutation, and the involvement of APC mutation. Typical 32 pilomatricomas were examined for beta-catenin expression by immunostaining. Genomic DNA was extracted, amplified and sequenced from 23 pilomaticomas with nuclear beta-catenin staining and 4 pilomaticomas without nuclear beta-catenin staining. Mutations of beta-catenin gene were confirmed by subcloning assay and restriction endonuclease assay. Using immunostaining, we found that 81% (26/32) of pilomatricomas displayed nuclear beta-catenin staining in basophilic cells. Sequence analysis revealed that 61% (14/23) contained mutations in exon 3 of beta-catenin. However, no mutations were detected in 4 pilomaticomas without beta-catenin nuclear staining. Detected mutations were adjacent to or abolished well-known regulatory phosphorylation sites of beta-catenin. APC gene mutations were not detected in 27 pilomatricomas with/without beta-catenin nuclear staining. These results confirmed that beta-catenin mutation and its nuclear localization are frequent causes of Wnt signaling pathway activation and suggested that beta-catenin activation mutations contribute to tumorigenesis of pilomatricomas.

Identification of MEKK2/3 serine phosphorylation site targeted by the Toll-like receptor and stress pathways

Members of the mitogen-activated protein kinase kinase kinase (MAP3K) family are crucial for the Toll-like receptor (TLR) signaling and cellular stress responses. However, the molecular mechanisms underlying the TLR- and cellular stress-mediated MAP3K activation remain largely unknown. In this study, we identified a key regulatory phosphorylation site, serine 519 and serine 526, in MAP3K MEKK2 and MEKK3, respectively. Mutation of this serine to an alanine severely impaired MEKK2/3 activation. We generated an anti-p-MEKK2/3 antibody and used this antibody to demonstrate that lipopolysaccharide induced MEKK2 and MEKK3 phosphorylation on their regulatory serine. We found that the serine phosphorylation was crucial for TLR-induced interleukin 6 production and this process is regulated by TRAF6, a key adaptor molecule for the TLR pathway. We further demonstrated that many, but not all, MAPK agonists induced the regulatory serine phosphorylation, suggesting an involvement of different MAP3Ks in activation of the MAPK cascades leading to different cellular responses. In conclusion, this study reveals a novel molecular mechanism for MEKK2/3 activation by the TLR and cellular stress pathways.

Bioactive small molecules reveal antagonism between the integrated stress response and sterol-regulated gene expression

Phosphorylation of translation initiation factor 2alpha (eIF2alpha) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2alpha. Furthermore, induction of the ISR by an artificial drug-activated eIF2alpha kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress.

The Phosphorylation of Ser318 of Insulin Receptor Substrate 1 Is Not per se Inhibitory in Skeletal Muscle Cells but Is Necessary to Trigger the Attenuation of the Insulin-stimulated Signal

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.