Induction of heme oxygenase-1 in the rat brain by kainic acid-mediated excitotoxicity: the dissociation of mRNA and protein expression in hippocampus.

Heme oxygenase-1 (HO-1) is induced under various stresses. Here we report the induction and localization of HO-1 in the rat brain by intraperitoneal administration of kainic acid (KA). Both mRNA and protein of HO-1 were markedly induced by KA treatment, and each maximal induction was observed 24 h after KA administration. In situ hybridization analysis showed that HO-1 mRNA appeared predominantly in glial cells, and confined neurons were positive in the cerebral cortex, basal ganglia, and hippocampal pyramidal cell layer. Immunohistochemical analysis showed that the positive cells in the cerebral cortex and hippocampus were mainly astrocytes and microglia, whereas neurons in the basal ganglia showed intense immunoreactivity. We also demonstrate the dissociation between HO-1 mRNA and protein level in the hippocampal pyramidal neurons, which is known to be vulnerable against excitotoxicity, and discuss the correlation between this dissociation and the vulnerability of hippocampal pyramidal neurons.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Loss of nuclear activity of the FBXO7 protein in patients with parkinsonian-pyramidal syndrome (PARK15)

Mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, an autosomal recessive neurodegenerative disease presenting with severe levodopa-responsive parkinsonism and pyramidal disturbances. Understanding the PARK15 pathogenesis might thus provide clues on the mechanisms of maintenance of brain dopaminergic neurons, the same which are lost in Parkinson's disease. The protein(s) encoded by FBXO7 remain very poorly characterized. Here, we show that two protein isoforms are expressed from the FBXO7 gene in normal human cells. The isoform 1 is more abundant, particularly in primary skin fibroblasts. Both isoforms are undetectable in cell lines from the PARK15 patient of an Italian family; the isoform 1 is undetectable and the isoform 2 is severely decreased in the patients from a Dutch PARK15 family. In human cell lines and mouse primary neurons, the endogenous or over-expressed, wild type FBXO7 isoform 1 displays mostly a diffuse nuclear localization. An intact N-terminus is needed for the nuclear FBXO7 localization, as N-terminal modification by PARK15-linked missense mutation, or N-terminus tag leads to cytoplasmic mislocalization. Furthermore, the N-terminus of wild type FBXO7 (but not of mutant FBXO7) is able to confer nuclear localization to profilin (a cytoplasmic protein). Our data also suggest that overexpressed mutant FBXO7 proteins (T22M, R378G and R498X) have decreased stability compared to their wild type counterpart. In human brain, FBXO7 immunoreactivity was highest in the nuclei of neurons throughout the cerebral cortex, intermediate in the globus pallidum and the substantia nigra, and lowest in the hippocampus and cerebellum. In conclusion, the common cellular abnormality found in the PARK15 patients from the Dutch and Italian families is the depletion of the FBXO7 isoform 1, which normally localizes in the cell nucleus. The activity of FBXO7 in the nucleus appears therefore crucial for the maintenance of brain neurons and the pathogenesis of PARK15.     

Posttranslational modification and cell type-specific degradation of varicella-zoster virus ORF29p

The ORF29 gene of varicella-zoster virus encodes a single-stranded DNA binding protein that is predominantly nuclear during lytic infection but appears to be restricted to the cytoplasm of latently infected neurons. Following reactivation, ORF29p accumulates in the nuclei of neurons, suggesting that its confinement to the cytosol may be critical for maintaining quiescence. When autonomously expressed, ORF29p accumulates in the nuclei of fibroblasts and the cytoplasm of cells (guinea pig enteric neurons) and cell lines (U373MG) of neuronal origin. Inhibition of the 26S proteasome redirects the accumulation of ORF29p to the nucleus in cells of neuronal origin. Here, we show that ORF29p is ubiquitinated and sumoylated in 293T cells and subsequently degraded from the N terminus. Ubiquitinated ORF29p accumulates in both the nuclei and the cytoplasm of fibroblasts, but degradation products are seen primarily in the cytoplasm. Modification and degradation of ORF29p occurs in 293T, U373MG, and MeWo cells. Therefore, these processes are ubiquitous; however, the robustness of the degradation process is cell type specific. The proteasome-mediated mechanism of nuclear exclusion in U373MG cells is an active process that is not specific for the endogenous ORF29p nuclear localization signal but can be saturated by protein stabilization or overexpression, which leads to nuclear accumulation of ORF29p. The evidence for ORF29p ubiquitination and previous data regarding the effect of proteasome inhibitors on the abundance and distribution of ORF29p implicate the 26S proteasome in influencing the protein's cell type-specific localization.     

βAmyloid蛋白在恒河猴脑组织中的形态学特点

目的观察βamyloid蛋白在不同年龄的恒河猴脑中的表达及其组织学和细胞超微结构水平的分布特点。方法分别取脑组织额叶、海马、颞叶和顶叶做免疫组化,观察βamyloid蛋白在组织学上的分布特点及与年龄的关系;选用23岁恒河猴一只,灌注后取上述部位做免疫电镜,观察βamyloid蛋白在细胞超微结构水平的分布特点。结果免疫组化染色可观察到年轻猴的神经细胞和胶质细胞中有少量的Aβ40颗粒,以额叶和海马居多。在老年猴脑中Aβ40常聚集成团状斑块。年轻猴脑细胞内未见明显的颗粒状Aβ42,而老年猴的额叶有多量的Aβ42细胞外散在斑块,神经细胞和胶质细胞内也见有Aβ42颗粒状沉积。免疫电镜可观察到Aβ40的胶体金颗粒大部分存在于老年猴神经细胞细胞质中,在细胞间质和小胶质细胞中也可见少量的胶体金颗粒,多见于额叶;标记Aβ42的胶体金颗粒也是多见于神经细胞中,在小胶质细胞中少量存在,同时在颞叶的神经纤维束中也可见Aβ42标记的胶体金颗粒。结论Aβ42是恒河猴老年斑的主要成分,并在其形成过程中起到重要作用。     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

A high affinity thyroid hormone binding protein in the cytosol of embryonic rat brain cells in primary cultures

A thyroid hormone binding protein(s) has been characterized in the cytosol of fetal rat brain cells in primary cultures. This protein is closely related to the one described in brain supernatants with respect to its electrophoretic mobility, binding kinetic parameters and estimated molecular weight (65 000 daltons). However, in contrast to the brain cytosolic binding protein, two classes of affinity sites for triiodothyronine (T3) and thyroxine (T4) have been demonstrated: a high affinity site (KA = 1.2-3.7(3) X 10(9) M-1 for T3 and KA = 3.7-5 X 10(8) M-1 for T4) and a low affinity site (KA = 0.8-1.4 X 10(8) M-1 for T3 and 1.6-2.9 X 10(7) M-1 for T4). The results are discussed with respect to their cellular significance.     

Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.     

The transport mechanism of metallothionein is different from that of classical NLS-bearing protein

A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS-mediated nuclear pore targeting is performed by using the cytosolic factors, importin alpha and importin beta, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS-mediated substrates. In digitonin-permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS-conjugated allophycocyanin (APC-NLS). Under ATP-free conditions, nuclear rim-binding was observed in both transport substrates. Rim-binding of labeled MT was competitively inhibited by the addition of an excess amount of unlabeled MT. Different elution profiles were observed for the localization-promoting activities of MT in the cytosol compared to those of APC-NLS. Furthermore, nuclear localization of MT was determined to be a wheat germ agglutinin-insensitive, GTPgammaS-sensitive, and anti-Ran antibody-sensitive process. Green fluorescent protein-metallothionein (GFP-MT) fusion protein was also localized in the nucleus in the stable transformant of CHL-IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization.     

Insulin-like growth factor-1 inhibits STS-induced cell death and increases functional recovery of in vitro differentiated neurons

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and down-regulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17,1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude without giving rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to 100% after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.     

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnβ1

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.     

Levetiracetam protects against kainic acid-induced toxicity

We investigated the Levetiracetam (LVT) ability to protect the brain against kainic acid (KA) induced neurotoxicity. Brain injury was induced by intraperitoneal administration of KA (10 mg/kg). Sham brain injury rats were used as controls. Animals were randomized to receive either LVT (50 mg/kg) or its vehicle (1 ml/kg) 30 min. before KA administration. Animals were sacrificed 6 hours after KA injection to measure brain malonildialdehyde (MDA), glutathione levels (GSH) and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of LVT decreased significantly MDA in the cortex (KA + vehicle = 0.25 +/- 0.03 nmol/mg protein; KA + LVT = 0.13 +/- 0.01 nmol/mg protein; P < 0.005), and in the diencephalons (KA + vehicle = 1,01 +/- 0.2 nmol/mg protein; KA + LVT = 0,33 +/- 0,08 nmol/mg protein; P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 5 +/- 1 micromol/g protein; KA + LVT = 15 +/- 2 micromol/g protein; P < 0.005) and diencephalons (KA + vehicle = 9 +/- 0.8 micromol/g protein; KA + LVT = 13 +/- 0.3 micromol/g protein; P < 0.05), reduced brain IL-1beta mRNA and markedly controlled seizures. Histological analysis showed a reduction of cell damage in LVT treated samples. The present data indicate that LVT displays neuro-protective effects against KA induced brain toxicity and suggest that these effects are mediated, at least in part, by inhibition of lipid peroxidation.     

Sumoylation of Daxx regulates IFN-induced growth suppression of B lymphocytes and the hormone receptor-mediated transactivation

Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.     

The effect of melatonin on lipid peroxidation and nitrite/nitrate levels, and on superoxide dismutase and catalase activities in kainic acid-induced injury

Kainic acid (KA) initiates neuronal injury and death by inducing oxidative stress and nitric oxide release from various regions of the brain. It was recently shown that melatonin has free radical-scavenging action and may protect against kainate-induced toxicity. In order to assess the possible supportive effect of melatonin treatment in KA-induced injury in the rat brain cortex, we determined malondialdehyde (MDA) levels as an index of lipid peroxidation, and assessed the activities of catalase (CAT) and superoxide dismutase (SOD) and the levels of nitrite/nitrate 35 male rats were divided into five groups, each receiving a different intraperitoneal treatment: saline solution (0.2 ml), kainic acid (15 mg/kg), melatonin (20 mg/kg), KA then melatonin (each as above, 15 min apart), or melatonin then KA (each as above, 30 min apart). Administration of KA caused an about five-fold increase in the catalase activity and an increase in the SOD activity in the cortex relative to the activities for the controls. Treatment with melatonin 15 min after KA injection kept malondialdehyde levels and catalase and superoxide dismutase activities at the normal levels, and led to an increase in the levels of nitrite/nitrate. Our data suggests that melatonin treatment following KA administration has a protective effect on antioxidant enzyme activities and thus supports the role of melatonin and oxidative stress in the regulation of antioxidative enzyme activity.     

Influence of neural grafting on rat brain with damaged temporal cortex

This study investigated the feasibility and certain aspects of grafting nerve tissue from 15- and 21-day embryo rats into the left temporal rat cortex damaged by a solution (1 µg/1 µliter) of kainic acid (KA). After unilateral damage induced in the temporal cortex by KA, extensive lesions were found in a number of brain structures (the hippocampus, thalamus, etc.) removed from the KA application site; asymmetry between hemispheres was also revealed from the areas of cross-sections of these structures. In the presence of temporal cortex from 21-day embryos grafted into the lesioned area and setting up axonal connections with the host brain, damage to brain structures removed from the lesioned site was either prevented or substantially reduced. Asymmetry between hemispheres as gauged from the area of their cross sections was no longer present in brain with such grafts; moreover, grafts from 15-day embryos transplanted into a cortex lesioned by KA projected out onto the brain surface, growing and compressing the latter. The damaging action of KA on the host brain extended in the presence of these grafts. A viable graft located within the damaged area is thought to inactivate excitatory transmitters accumulating due to KA action, probably fulfilling the function of the damaged cortex in some measure once connections with the host brain have been set up.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 586–595, October–September, 1990.     

Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.     

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import [published erratum appears in J Cell Biol 1992 Jul;118(1):215]

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol. NBPs are phosphorylated and phosphatase treatment abolished NLS binding. The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells. Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP. In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope. Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus. Anti-NBP antibodies specifically inhibited the binding part of this import reaction. These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.     

Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry

We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.