14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

Nuclear localization of protein kinase U-alpha is regulated by 14-3-3.

14-3-3 proteins are intracellular, dimeric molecules that bind to and modify the activity of several signaling proteins. We used human 14-3-3zeta as a bait in the yeast two-hybrid system to screen a murine embryonic cDNA library. One interacting clone was found to encode the carboxyl terminus of a putative protein kinase. The coding sequence of the human form (protein kinase Ualpha, PKUalpha) of this protein kinase was found in GenBank(TM) on the basis of sequence homology. The two-hybrid clone was also highly homologous to TOUSLED, an Arabidopsis thaliana protein kinase that is required for normal flower and leaf development. PKUalpha has been found by coimmunoprecipitation to bind to 14-3-3zeta in vivo. Our confocal laser immunofluorescence microscopic experiments revealed that PKUalpha colocalizes with the cytoplasmic intermediate filament system of cultured fibroblasts in the G(1) phase of the cell cycle. PKUalpha is found in the perinuclear area of S phase cells and in the nucleus of late G(2) cells. Transfection of cells with a dominant negative form of 14-3-3eta promotes the nuclear localization of PKUalpha. These results suggest that the subcellular localization of PKUalpha is regulated, at least in part, by its association with 14-3-3.     

Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP

Among three different isoforms of non-muscle myosin heavy chains (NMMHCs), only NMMHCA is associated with inherited human disease, called MYH9 disorders, characterized by macrothrombocytopenia and characteristic granulocyte inclusions. Here targeted gene disruption was performed to understand fundamental as well as pathological role of the gene for NMMHCA, MYH9. Heterozygous intercrosses yielded no homozygous animals among 552 births, suggesting that MYH9 expression is required for embryonic development. In contrast, MYH9+/- mice were viable and fertile without gross anatomical, hematological, and nephrological abnormalities. Immunofluorescence analysis also showed the normal cytoplasmic distribution of NMMHCA. We further measured the auditory brainstem response and found two of six MYH9+/- mice had hearing losses, whereas the remaining four were comparable to wild-type mice. Such observation may parallel the diverse expression of Alport's manifestations of human individuals with MYH9 disorders and suggest the limited requirement of the gene for maintenance and function of specific organs.     

Protein kinase C mu is negatively regulated by 14-3-3 signal transduction proteins

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3tau isoform has been shown to associate with protein kinase C theta and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau interacts with protein kinase C mu (PKCmu), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCmu and 14-3-3tau can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCmu-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCmu deletion mutants, the 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau to PKCmu is significantly enhanced upon phorbol ester stimulation of PKCmu kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3tau is not a substrate of PKCmu. In contrast 14-3-3tau strongly down-regulates PKCmu kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCmu kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCmu in T cells.     

14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3zeta protein.     

14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

Background  

Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G₂,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade.     

Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals

Proteins with dibasic retention motifs are subject to retrograde transport to endoplasmic reticulum (ER) by COPI-coated vesicles. As forward transport requires escape from ER retention, general release mechanisms have been expected. Here, KCNK3 potassium channels are shown to bear two cytoplasmic trafficking motifs: an N-terminal dibasic site that binds beta-COP to hold channels in ER and a C-terminal "release" site that binds the ubiquitous intracellular regulator 14-3-3beta on a nonclassical motif in a phosphorylation-dependent fashion to suppress beta-COP binding and allow forward transport. The strategy appears to be common. The major histocompatibility antigen class II-associated invariant chain Iip35 exhibits dibasic retention, carries a release motif, and shows mutually exclusive binding of beta-COP and 14-3-3beta on adjacent N-terminal sites. Other retained proteins are demonstrated to carry functional 14-3-3beta release motifs.     

Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms

Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. The inhibition is reversed by Ca2+/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sites specific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought to be involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal of inhibition is not well understood. We have expressed His6-tagged fragments containing the sequence of the C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well as a variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed by high-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminal region of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescence resonance energy transfer between probes attached to cysteine residues (the endogenous C595 and the engineered C766) located in these two clusters revealed that the C-terminal region of CaD is elongated, but it becomes more compact when bound to actin. Binding of CaM restores the elongated conformation and facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment was phosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereas the other remained bound. Taken together, these results demonstrate that while both Ca2+/CaM and MAPK phosphorylation govern CaD's function via a conformational change, the regulatory mechanisms are different.     

BAD association with membranes is regulated by Raf kinases and association with 14-3-3 proteins

Using a set of specific kinase inhibitors we demonstrate that Raf kinases phosphorylate BAD at serines 112, 136 and 155 in vivo and in vitro. Exploring unexpected lipid binding properties of BAD we identified two lipid-binding domains located in its C-terminal part. Furthermore, we believe to have uncovered how phosphorylation-driven interaction with 14-3-3 regulates intracellular membrane localization of BAD. Observed activity of lipid-bound BAD as a membrane receptor for Bcl-XL opens new horizons in apoptosis research.     

Nedd4-2 binding to 14-3-3 modulates the accessibility of its catalytic site and WW domains

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.     

Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease

Background

Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson''s disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.

Methodology/Principal Findings

Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.

Conclusions/Significance

LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.     

Phosphatidylserine transport to the mitochondria is regulated by ubiquitination

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.     

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.     

Golgi-to-late endosome trafficking of the yeast pheromone processing enzyme Ste13p is regulated by a phosphorylation site in its cytosolic domain

This study addressed whether phosphorylation regulates trafficking of yeast membrane proteins that cycle between the trans-Golgi network (TGN) and endosomal system. The TGN membrane proteins A-ALP, a model protein containing the Ste13p cytosolic domain fused to alkaline phosphatase (ALP), and Kex2p were found to be phosphorylated in vivo. Mutation of the S13 residue on the cytosolic domain of A-ALP to Ala was found to block trafficking to the prevacuolar compartment (PVC), whereas a S13D mutation generated to mimic phosphorylation accelerated trafficking into the PVC. The S13 residue was shown by mass spectrometry to be phosphorylated. The rate of endoplasmic reticulum-to-Golgi transport of newly synthesized A(S13A)-ALP was indistinguishable from wild-type, indicating that the lack of transport of A(S13A)-ALP to the PVC was instead due to differences in Golgi/endosomal trafficking. The A(S13A)-ALP protein exhibited a TGN-like localization similar to that of wild-type A-ALP. Similarly, the S13A mutation in endogenous Ste13p did not reduce the extent of or longevity of its localization to the TGN as shown by alpha-factor processing assays. These results indicate that S13 phosphorylation is required for TGN-to-PVC trafficking of A-ALP and imply that phosphorylation of S13 may regulate recognition of A-ALP by vesicular trafficking machinery.     

Regulation of TSC2 by 14-3-3 binding

Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.     

Inhibition of chloride binding to the anion transport site by diethylpyrocarbonate modification of band 3

Summary The line widths of³⁵Cl^– nuclear magnetic resonances were used to measure chloride binding by Band 3. Since this procedure related directly to binding, the data obtained may be interpreted more unequivocally than affinities derived from kinetic data which could be related to either translocation or binding. Chloride binding to the active sites in Band 3 was assessed from that portion of the total line width which was sensitive to 4,4-dinitrostilbene-2,2-disulfonic acid. These sites appeared to be completely inhibited by treatment of erythrocyte membranes with diethylpyrocarbonate. This result is consistent with our previous observation that this reagent inhibits anion transport in resealed erythrocyte ghosts (Izuhara, Okubo & Hamasaki, 1989,Biochemistry 28:4725–4728). Hydroxylamine could not reverse the diethylpyrocarbonate inhibition of chloride binding to Band 3. The pH-dependence of diethylpyrocarbonate reactivity suggests that the modified residues may be those of histidine.