Phosphorylation Of Lamin Research Articles

Inactivation of Retinoblastoma Protein Does Not Overcome the Requirement for Human Cytomegalovirus UL97 in Lamina Disruption and Nuclear Egress

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

Hierarchical Determination of Nuclear Deformability by Lamin Isoforms During Adult Hematopoiesis: Implications in Blood Cell Trafficking

Abstract 1200Over the last few decades, it has become clear that different blood lineages have distinct deformability, and that some blood cell types become softer in maturation perhaps to facilitate trafficking from marrow through the endothelial barrier and into the circulation (Lichtman, NEJM, 1970; 283:943–8). However, any common molecular basis of this phenomenon remains unclear. Cellular deformability is determined by elasticity of the cortex and the nucleus, and the latter is dynamically regulated by changes in expression and organization of the lamins (Pajerowski… Discher, PNAS, 2007; 104:15619–24). While gene regulation of hematopoiesis has been extensively studied, roles of lamins in blood lineages are less well understood. To address this, we developed a novel protein isoform expression analysis algorithm, “mass spectrometry calibrated intracellular flow cytometry”, to quantify lamin stoichiometries in human hematopoietic stem cells and progenitors through different mature blood lineages. This approach reveals the hematopoietic lineage map of lamins, showing that lamin A varies by 4-fold, while the normally ‘constitutive' lamin B varies by 30-fold. During differentiation, lymphoid and myeloid lineages show decreased total lamin intensity and pliable nuclei as measured by micropipette aspiration, consistent with their ability to transmigrate into circulation. In contrast, megakaryocytes (MKs) remain in marrow because their polyploid nuclei are too large and rigid, as indicated by high lamin levels; this nuclear anchorage allows MKs to extend membrane projections into blood, where shear generates circulating platelets. Maturation of MKs is further regulated by serine phosphorylation of lamin A, since overexpression of a phospho-inactive mutant leads to increased polyploidization. Erythroid lineages share the same progenitor with MKs and migrate into blood as enucleated RBCs, because of high lamin A intensity relative to B in the progenitors and stiff chromatin. Consistent with this observation, microarray analysis of primary CD34+-derived cells indicates that some key erythroid genes are strongly correlated with lamin isoforms. Functional studies indicate that lamin A overexpression increases MK and erythroid differentiation by 2-fold, while the knockdown increases migration through pores by 2-fold. Surprisingly, increasing the lamin A to B ratio by lamin B1 knockdown decreases nuclear deformability by up to 50%, highlighting the importance of lamin isoform ratios, rather than absolute expression levels, in specifying nuclear rheology and hence traffickability of blood lineages. Finally, lamin A can be transcriptionally downregulated by retinoic acid by 2-fold, consistent with its well-established role in driving myeloid differentiation from progenitors. Together, the study suggests that nuclear deformability is hierarchically programmed by differential expression of the nucleoskeletal lamin A and B isoforms during hematopoietic differentiation, which in turn influence the ability of blood cells to migrate through marrow. Disclosures:No relevant conflicts of interest to declare.

The Role of Nuclear Matrix Proteins Binding to Matrix Attachment Regions (MARs) in Prostate Cancer Cell Differentiation

In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.

Nuclear Envelope Budding Enables Large Ribonucleoprotein Particle Export during Synaptic Wnt Signaling

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. We previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. Here we show that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules.

Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B Receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Herein, we use time‐lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin‐binding ability of LBR together with Aurora B activity contribute to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

Nuclear envelope dispersion triggered by deregulated Cdk5 precedes neuronal death

Nuclear fragmentation is a common feature in many neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we show that nuclear lamina dispersion is an early and irreversible trigger for cell death initiated by deregulated Cdk5, rather than a consequence of apoptosis. Cyclin-dependent kinase 5 (Cdk5) activity is significantly increased in AD and contributes to all three hallmarks: neurotoxic amyloid-β (Aβ), neurofibrillary tangles (NFT), and extensive cell death. Using Aβ and glutamate as the neurotoxic stimuli, we show that deregulated Cdk5 induces nuclear lamina dispersion by direct phosphorylation of lamin A and lamin B1 in neuronal cells and primary cortical neurons. Phosphorylation-resistant mutants of lamins confer resistance to nuclear dispersion and cell death on neurotoxic stimulation, highlighting this as a major mechanism for neuronal death. Rapid alteration of lamin localization pattern and nuclear membrane change are further supported by in vivo data using an AD mouse model. After p25 induction, the pattern of lamin localization was significantly altered, preceding neuronal death, suggesting that it is an early pathological event in p25-inducible transgenic mice. Importantly, lamin dispersion is coupled with Cdk5 nuclear localization, which is highly neurotoxic. Inhibition of nuclear dispersion rescues neuronal cells from cell death, underscoring the significance of this event to Cdk5-mediated neurotoxicity.

Specific phosphorylation of Ser458 of A-type lamins in LMNA-associated myopathy patients

Mutations in LMNA, which encodes A-type nuclear lamins, cause various human diseases, including myopathy, cardiomyopathy, lipodystrophy and progeria syndrome. To date, little is known about how mutations in a single gene cause a wide variety of diseases. Here, by characterizing an antibody that specifically recognizes the phosphorylation of Ser458 of A-type lamins, we uncover findings that might contribute to our understanding of laminopathies. This antibody only reacts with nuclei in muscle biopsies from myopathy patients with mutations in the Ig-fold motif of A-type lamins. Ser458 phosphorylation is not seen in muscles from control patients or patients with any other neuromuscular diseases. In vitro analysis confirmed that only lamin A mutants associated with myopathy induce phosphorylation of Ser458, whereas lipodystrophy- or progeria-associated mutants do not. We also found that Akt1 directly phosphorylates Ser458 of lamin A with myopathy-related mutations in vitro. These results suggest that Ser458 phosphorylation of A-type lamins correlates with striated muscle laminopathies; this might be useful for the early diagnosis of LMNA-associated myopathies. We propose that disease-specific phosphorylation of A-type lamins by Akt1 contributes to myopathy caused by LMNA mutations.

Specific phosphorylation of Ser458 of A-type lamins inLMNA-associated myopathy patients

Mutations in LMNA, which encodes A-type nuclear lamins, cause various human diseases, including myopathy, cardiomyopathy, lipodystrophy and progeria syndrome. To date, little is known about how mutations in a single gene cause a wide variety of diseases. Here, by characterizing an antibody that specifically recognizes the phosphorylation of Ser458 of A-type lamins, we uncover findings that might contribute to our understanding of laminopathies. This antibody only reacts with nuclei in muscle biopsies from myopathy patients with mutations in the Ig-fold motif of A-type lamins. Ser458 phosphorylation is not seen in muscles from control patients or patients with any other neuromuscular diseases. In vitro analysis confirmed that only lamin A mutants associated with myopathy induce phosphorylation of Ser458, whereas lipodystrophy- or progeria-associated mutants do not. We also found that Akt1 directly phosphorylates Ser458 of lamin A with myopathy-related mutations in vitro. These results suggest that Ser458 phosphorylation of A-type lamins correlates with striated muscle laminopathies; this might be useful for the early diagnosis of LMNA-associated myopathies. We propose that disease-specific phosphorylation of A-type lamins by Akt1 contributes to myopathy caused by LMNA mutations.

Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

BackgroundThe eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.Methodology and Principal FindingsWe replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.Conclusions and SignificanceK6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

BackgroundDuring fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development.Methodology/Principal FindingsInhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition.Conclusions/SignificanceOur findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.

Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.

Role of the Endoplasmic Reticulum Chaperone BiP, SUN Domain Proteins, and Dynein in Altering Nuclear Morphology during Human Cytomegalovirus Infection

The process of assembly and egress of human cytomegalovirus (HCMV) virions requires significant morphological alterations of the nuclear and cytoplasmic architecture. In the studies presented we show that the nuclear periphery is dramatically altered, especially near the cytoplasmic assembly compartment, where the nuclear lamina is specifically rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. In addition, the tethering of the inner and outer nuclear membranes is lost during infection due to a decrease in levels of the SUN domain proteins. We previously demonstrated that the endoplasmic reticulum protein BiP functions as a component of the assembly compartment and disruption of BiP causes the loss of assembly compartment integrity. In this study we show that the depletion of BiP, and the loss of assembly compartment integrity, results in the loss of virally induced lamina rearrangement and morphology of the nucleus that is characteristic of HCMV infection. BiP functions in lamina rearrangement through its ability to affect lamin phosphorylation. Depletion of BiP and disruption of the assembly compartment result in the loss of lamin phosphorylation. The dependency of lamin phosphorylation on BiP correlates with an interaction between BiP and UL50. Finally, we confirm previous data (S. V. Indran, M. E. Ballestas, and W. J. Britt, J. Virol. 84:3162-3177, 2010) suggesting an involvement of dynein in assembly compartment formation and extend this observation by showing that when dynein is inhibited, the nuclear morphology characteristic of an HCMV infection is lost. Our data suggest a highly integrated assembly-egress continuum.

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser(22) generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells.

Simian Virus 40 T/t Antigens and Lamin A/C Small Interfering RNA Rescue the Phenotype of an Epstein-Barr Virus Protein Kinase (BGLF4) Mutant

The Epstein-Barr virus (EBV)-encoded viral protein kinase, EBV-PK (the BGLF4 gene product), is required for efficient nuclear viral egress in 293 cells. However, since EBV-PK phosphorylates a number of different viral and cellular proteins (including lamin A/C), the relative importance of each target during lytic viral replication remains unclear. We show here that an EBV PK mutant (PKmut; containing stop codons at residues 1 and 5 in EBV-PK) is highly defective for release of infectious virus from 293 cells but not 293T cells. Furthermore, the phenotype of the PKmut in 293 cells is substantially reversed by expression of the simian virus 40 (SV40) large (T) and small (t) T antigens. Efficient rescue requires the presence of both SV40 T/t proteins. We show that 293T cells have a much higher level of constitutive lamin A/C phosphorylation than do 293 cells over residues (S22 and S392) that promote phosphorylation-dependent nuclear disassembly and that both large T and small t contribute to enhanced lamin A/C phosphorylation. Finally, we demonstrate that knockdown of lamin A/C expression using small interfering RNA also rescues the PKmut phenotype in 293 cells. These results suggest that essential roles of EBV-PK during lytic viral replication include the phosphorylation and dispersion of lamin A/C.

Nuclear Lamins in Cell Regulation and Disease

The nuclear lamins are type V intermediate filament proteins that form meshworks at the inner aspect of the nuclear envelope and are also present throughout the nuclear interior. Through these meshwork structures, lamins regulate the shape, size, and mechanical properties of the nucleus. During the last 25 years, the Goldman laboratory has studied the organization and dynamic properties of the lamins in the nucleus. These studies have characterized the role of lamin phosphorylation in nuclear lamina assembly and disassembly during mitosis. Furthermore, our studies have demonstrated a role for the lamins in chromatin modification and epigenetics, transcription, and DNA replication. Recently, the discovery of numerous mutations in the gene encoding A-type lamins causing the collection of diseases known as laminopathies has provided new insights into the roles of lamins in cellular regulation and differentiation.

Phosphorylation of GFAP and lamin B is an early event in reactive astrocytes and may be stimulated by cAMP

Mammalian astrocytes respond to injury by hypertrophy and cell division. Changes in the intermediate filament (IF) cytoskeleton and the lamin nucleo‐skeleton proceeds by phosphorylation of IF subunits, solubilization and reformation after cell division. Monoclonal AB J1‐31 recognizes a phosphoepitope found on GFAP and lamin B in astrocytes and thus is useful for exploring pathways to the reactive state. F98 (a.k.a. 9L) glioblastoma cells have been used to model gliosis in vitro. To test the hypothesis that a cAMP mediated pathway is involved, we added forskolin to confluent cultures of F98 cells and assayed for phosphorylation of lamin B by measuring binding of mAB J1‐31. Forskolin caused a 2X increase in mAB J1‐31 binding to nuclear structures also labeled by anti‐lamin B, and a lesser increase in cytoplasmic labeling of filaments also labeled by anti‐GFAP. Binding of anti‐lamin B and anti‐GFAP to their respective antigens was unaffected by forskolin. Inhibitors of PKA (H89) were not effective in preventing the forskolin induced phosphorylation of lamin B and GFAP, but inhibitors of Ca+2 influx (verapamil) and protein synthesis (cycloheximide) did significantly reduce the effect of forskolin.

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12 Cells

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.

U S 3 of Herpes Simplex Virus Type 1 Encodes a Promiscuous Protein Kinase That Phosphorylates and Alters Localization of Lamin A/C in Infected Cells

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.