Regulated Shuttling of Slit-Robo-GTPase Activating Proteins Between Nucleus and Cytoplasm During Brain Development

(1) A Little information exists on the distribution of Slit-Robo-GTPase activating proteins (srGAPs), particularly about their intracellular locations, which may provide further clues to their functions. The purpose of this study is to elucidate the expression patterns of the three srGAPs in wild-type rat brains at adult and various developmental stages, and in the cultured cortical neurons. (2) Immunohistochemical method was applied to detect the distribution and localization of the srGAPs in the normal rat brains at adult and various developmental stages, and in the cultured cortical neurons using the rabbit polyclonal antibodies. (3) Immunohistochemical analysis demonstrated that the three srGAPs were mainly expressed in neurons throughout the brain. More importantly, srGAPs translocated during development by a highly regulated shuttling process between the nucleus and the cytoplasm of neurons and their expression patterns were not overlapping. In cultured cortical neurons srGAPs were found in equal amounts in the cytoplasm, nucleus, in neurites, and growth cones. When neurons were maintained in vitro for longer time, the amount of srGAPs in the nucleus strongly increased. (4) These results suggest that srGAPs are not only involved in the regulation of the Slit-Robo signal transduction, but also in neuronal development and that the translocation of srGAPs is important for their functions. Qin Yao and Wei-Lin Jin are contributed equally to this work     

多头绒泡菌类p34cdc2蛋白的定位研究

目前关于动物和酵母细胞中p34cdc2的定位研究结果尚存在分歧,而关于该蛋白在植物细胞中的定位尚不清楚.以多头绒泡菌( Physarum polycephalum )S期、G2早期、G2中期、G2晚期、前期、中期和后末期的原质团和细胞核为材料进行免疫印迹,发现原质团和细胞核都含有一种分子量约34 kD的类p34cdc2蛋白,该蛋白在原质团和细胞核中的含量在整个细胞周期进程中基本保持稳定.以抗p34cdc2单克隆抗体为探针的免疫电镜结果显示,类p34cdc2蛋白既分布于细胞核也分布于细胞质中,在细胞核中主要与染色体和核仁结合.经抗p34cdc2单克隆抗体处理后,多头绒泡菌的有丝分裂启始迟滞约2 h.结果表明,多头绒泡菌类p34cdc2蛋白存在于细胞核和细胞质中,与细胞有丝分裂密切相关,其含量在细胞周期进程中基本保持稳定.     

JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.     

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.     

The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10⁻⁷ cm²/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/D_o) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [R_G] 40–300 Å). The fraction of mobile FITC-dextran molecules (f_mob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for R_G < 200 Å, but decreased to <0.5 for R_G > 300 Å. The independence of D/D_o on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/D_o, indicating that microcompartments, if present, are of submicron size. Measurements of D/D_o and f_mob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low f_mob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/D_o was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (R_G 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.     

A Novel cdc2-Related Protein Kinase Expressed in the Nervous System

Abstract: We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34^cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of ～58–60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34^cdc2 and cdk5.     

MAP and cdc2 Kinase Activities at Germinal Vesicle Breakdown inChaetopterus

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

PINK1 Kinase Catalytic Activity Is Regulated by Phosphorylation on Serines 228 and 402

Mutations in the PINK1 gene cause early-onset recessive Parkinson disease. PINK1 is a mitochondrially targeted kinase that regulates multiple aspects of mitochondrial biology, from oxidative phosphorylation to mitochondrial clearance. PINK1 itself is also phosphorylated, and this might be linked to the regulation of its multiple activities. Here we systematically analyze four previously identified phosphorylation sites in PINK1 for their role in autophosphorylation, substrate phosphorylation, and mitophagy. Our data indicate that two of these sites, Ser-228 and Ser-402, are autophosphorylated on truncated PINK1 but not on full-length PINK1, suggesting that the N terminus has an inhibitory effect on phosphorylation. We furthermore establish that phosphorylation of these PINK1 residues regulates the phosphorylation of the substrates Parkin and Ubiquitin. Especially Ser-402 phosphorylation appears to be important for PINK1 function because it is involved in Parkin recruitment and the induction of mitophagy. Finally, we identify Thr-313 as a residue that is critical for PINK1 catalytic activity, but, in contrast to previous reports, we find no evidence that this activity is regulated by phosphorylation. These data clarify the regulation of PINK1 through multisite phosphorylation.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.