Phosphorylation of the major Drosophila lamin in vivo: site identification during both M-phase (meiosis) and interphase by electrospray ionization tandem mass spectrometry

Phosphorylation can have profound effects on the properties of nuclear lamins. For instance, phosphorylation of specific sites on mammalian lamins drastically alters their propensity to polymerize. Relatively little is known about the effects of phosphorylation during interphase and about phosphorylation of invertebrate nuclear lamins. Here, using electrospray ionization tandem mass spectrometry, we determined the phosphorylation sites of both interphase and M-phase isoforms of nuclear lamin Dm from Drosophila melanogaster. Interphase lamins are phosphorylated at three sites: two of these sites (Ser25 and a site located between residues 430 and 438) flank the alpha-helical rod domain, whereas the third site (Ser595) is located close to the C-terminus. The M-phase lamin isoform is phosphorylated predominantly at Ser45, a residue contained within a sequence matching the consensus site for phosphorylation by cdc2 kinase. Our study confirms the important role in vivo for cdc2 kinase in M-phase disassembly of nuclear lamins and provides the basis for understanding Drosophila lamin phosphorylation during interphase.     

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G₂ checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G₂ arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G₂ arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G₂ checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G₂ checkpoint, phospho-Bcl-xL(Ser62) stabilizes G₂ arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.     

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G₂ checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G₂ arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G₂ arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G₂ checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G₂ checkpoint, phospho-Bcl-xL(Ser62) stabilizes G₂ arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells

MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.     

Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2.

Members of the mitogen-activated protein (MAP) kinase family are implicated in mediating entry of cells into the cell cycle, as well as passage through meiotic M phase. These kinases have attracted much interest because their activation involves phosphorylation on both tyrosine and threonine residues, but little is known about their physiological targets. In this study, two distinct members of the MAP kinase family (p44mpk and p42mapk) are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16. Moreover, these MAP kinases cause depolymerization of in-vitro-assembled longitudinal lamin head-to-tail polymers. Ser16 was previously shown to be phosphorylated during mitosis in vivo, and to be a target of the mitotic protein kinase p34cdc2 in vitro. Accordingly, lamins were proposed to be direct in vivo substrates of p34cdc2. This proposal is supported by quantitative analyses indicating that lamin B2, when assayed in vitro, is a substantially better substrate for p34cdc2 than for MAP kinases. Nevertheless, a physiological role of MAP kinases in lamin phosphorylation is not excluded. The observation that members of the MAP kinase family display sequence specificities overlapping that of p34cdc2 raises the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrates of MAP kinases.     

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA,but interacts with its nuclear envelope partners

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.     

Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.

The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.     

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.     

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.     

A role for the phosphorylation of hRad9 in checkpoint signaling

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.     

A Phos‐tag SDS‐PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1

MEK1, an essential component of the mitogen‐activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS‐based phosphoproteomic methods. By using Phos‐tag SDS‐PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos‐tag‐based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time‐course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti‐HaloTag antibody, we analyzed a HaloTag‐fused MEK1 protein and 12 potential phosphorylation‐site‐directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser‐218 and Ser‐222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor‐specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK‐733, PD98059, and U0126.