Interactive Fly, Drosophila



Lamina-associated polypeptides: proteins that interact with Lamins

Lamina-associated polypeptides (LAPs) 1A, 1B, 1C, and 2 are integral membrane proteins of the nuclear envelope associated with the nuclear lamina. LAPs 1A and 1B specifically bind to both lamins A and C and lamin B1, while LAP 2 associates only with lamin B1. LAP 2 also binds to mitotic chromosomes. The LAPs are phosphorylated during mitosis, and phosphorylation of LAP 2 by mitotic cytosol inhibits its binding to both lamin B1 and chromosomes. During late anaphase, LAP 2 associates with chromosomes prior to assembly of most lamins. Together, these data suggest that LAP 2 may have a key role in initial events of nuclear envelope reassembly, and that both LAP 2 and LAP 1 may be involved in attaching lamins to the nuclear envelope (Foisner, 1993).

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds directly to both lamin B1 and chromosomes in a mitotic phosphorylation-regulated manner. The biochemical and physiological properties of LAP2 suggest an important role in nuclear envelope reassembly at the end of mitosis and/or anchoring of the nuclear lamina and interphase chromosomes to the nuclear envelope. The cDNA cloning of LAP2 is described as well as the characterization of its membrane topology and targeting to the nuclear envelope. The LAP2 cDNA sequence predicts a protein of 452 amino acids, containing a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane-spanning sequence at residues 410-433. Immunogold localization of an LAP2 epitope in isolated nuclear envelopes indicates that the large amino-terminal hydrophilic domain (residues 1-409) is exposed to the nucleoplasm. By expressing deletion mutants of LAP2 in cultured cells, multiple regions in its nucleoplasmic domain have been identified that promote localization at the nuclear envelope. These data suggest that targeting of LAP2 to the nuclear envelope is mediated by cooperative interactions with multiple binding sites at the inner nuclear membrane (Furukawa, 1995).

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. Microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it is also caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298-373) but not by the chromatin-binding domain (residues 1-88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, these results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume (Yang, 1997b).

The in situ organization and nearest neighbours of the 'lamina-associated polypeptide-1' (LAP1), a type II membrane protein and a major constituent of the mammalian nuclear envelope have been examined. During interphase, LAP1 forms multimeric assemblies that are suspended in the inner nuclear membrane and are specifically associated with B-type lamins. The LAP1-lamin B complex is distinct from analogous complexes formed by the 'lamina-associated polypeptide-2' (LAP2), another inner nuclear membrane protein, and includes a protein kinase. Upon nuclear envelope breakdown, LAP1 partitions with mitotic vesicles that carry nuclear lamin B. The LAP1 vesicles can be distinguished from fragments of the nuclear envelope containing LAP2 and exhibit a striking co-alignment with spindle microtubules. These observations suggest that the inner nuclear membrane comprises discrete territories that accommodate specific integral membrane proteins and are differentially disassembled during mitosis (Maison, 1997).

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis (Collas, 1999).

Lamin B receptor (LBR), a Lamin interacting protein

At the nuclear envelope in higher eukaryotic cells, the nuclear lamina and the heterochromatin are adjacent to the inner nuclear membrane; their attachment is presumably mediated by integral membrane proteins. In a yeast two-hybrid screen, the nucleoplasmic domain of lamin B receptor (LBR), an integral protein of the inner nuclear membrane, associates with two human polypeptides homologous to Drosophila HP1, a heterochromatin protein involved in position-effect variegation. LBR binds to B-type lamins and to double-stranded DNA. Human LBR has a nucleoplasmic, amino terminal domain of 208 amino acids followed by eight putative transmembrane segments. LBR fusion proteins bind to HP1 proteins synthesized by in vitro translation and present in cell lysates. Antibodies against LBR also co-immunoprecipitated HP1 proteins from cell extracts. LBR can interact with chromodomain proteins that are highly conserved in eukaryotic species and may function in the attachment of heterochromatin to the inner nuclear membrane in cells. LBR undergoes phosphorylation catalyzed by p34cdc2 protein kinase in mitosis when the inner nuclear membrane breaks down into vesicles that dissociate from the lamina and the chromatin. It is phosphorylated by different protein kinases in interphase when the membrane is associated with these structures. Phosphorylation of LBR and/or HP1 proteins may therefore be responsible for some of the alterations in chromatin organization and nuclear structure that occur at various times during the cell cycle (Ye, 1996).

Employing avian erythrocytes, a multimeric complex has been isolated. This consists of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. p18 is an integral membrane protein specific to the erythrocyte nuclear envelope that binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane (Simos, 1996).

The primary structure of human LBR, an integral protein of the nuclear envelope inner membrane is 68% identical to the chicken lamin B receptor and has a basic nucleoplasmic amino-terminal domain of 208 amino acids followed by a hydrophobic domain containing eight putative transmembrane segments. The amino-terminal domain contains a Ser-Arg-rich stretch and consensus sites for phosphorylation by protein kinase A and p34cdc2 protein kinase. A fusion protein containing the amino-terminal domain of human LBR is recognized by autoantibodies from patients with primary biliary cirrhosis, and these serum antibodies label the nuclear envelope when examined by immunofluorescence microscopy. The LBR amino-terminal domain precipitates lamin B from nuclear extracts and retards the migration of double-stranded DNA subjected to agarose gel electrophoresis. The amino-terminal domain of LBR also associates with DNA; the stretch between amino acids 71 and 100, which contains the Ser-Arg-rich stretch, is necessary for DNA binding. These results demonstrate that LBR is conserved among vertebrate species and that its nucleoplasmic domain can potentially mediate the interaction of both the nuclear lamina and the chromatin with the inner nuclear membrane (Ye, 1994).

A nuclear localization signal (NLS) binding protein, NBP60, has been characterized in rat liver nuclear envelopes. Rat NBP60 has 620 amino acids, is a homolog of lamin B receptor (LBR), and is 79 and 63% identical in amino acids to human and chicken LBR, respectively. The stretch comprising amino acids 1 to 89, which contains a Ser-Arg rich region (RS region), binds to nucleoplasmin; the binding is inhibited by a common NLS-peptide. These results suggested that the amino-terminal domain of LBR contains an NLS-binding site. The stretch comprising amino acids 1 to 53, which does not contain the RS region or the predicted DNA-binding site, binds to Xenopus laevis sperm chromatin (Kawahire, 1997).

Morphological studies have established that peripheral heterochromatin is closely associated with the nuclear envelope. The tight coupling of the two structures has been attributed to nuclear lamins and lamin-associated proteins; however, it remains to be determined which of these elements are essential and which play an auxiliary role in nuclear envelope-chromatin interactions. A model system was used: in vitro reconstituted vesicles assembled from octyl glucoside-solubilized nuclear envelopes. The principal chromatin anchorage site at the nuclear envelope is the lamin B receptor (LBR), a ubiquitous integral protein of the inner nuclear membrane. Consistent with this interpretation, purified LBR binds directly to chromatin fragments and decorates the surface of chromosomes in a distinctive banding pattern (Pyrpasopoulou, 1996).

An integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) that contribute the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. It is suggested that p56 is a sea urchin LBR homolog that targets membranes to chromatin and later anchors the membrane to the lamina (Collas, 1996).

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, phosphopeptide mapping was performed using in vitro and in vivo 32P-labeled LBR; a series of recombinant proteins and synthetic peptides were analyzed. LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34(cdc2) protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34(cdc2) mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly (Nikolakaki, 1997).

Lamin and apoptosis

In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity parallels chromatin fragmentation and the appearance of small apoptotic cells. (For more information on apoptosis, see Reaper). The elevation of nuclear proteolytic activity is accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurs within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generates a fragment of 46 kDa consistent with the central rod domain of the protein, is also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation does not change as a result of the dexamethasone treatment and the lamina does not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occur throughout the nuclei; solubilization of lamina is not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis (Weaver, 1996).

CrmA, a poxvirus gene product with a serpin-like structure, blocks a variety of apoptotic death events in cultured cells. Based on the ability of CrmA to inhibit the interleukin-1beta converting enzyme in vitro, it has been speculated that interleukin-1beta converting enzyme-related proteases (caspases) essential for apoptosis are the cellular targets of CrmA (See Drosophila Death caspase). Rabbitpox virus CrmA/SPI-2 inhibits the cleavage of lamin A mediated by a caspase in a cell-free system of apoptosis. In the presence of CrmA/SPI-2, nuclear apoptosis in vitro is blocked at an intermediate stage after collapse of the chromatin against the nuclear periphery and before nuclear shrinkage and disintegration into apoptotic body-like fragments. One of five caspases active in the extracts is inhibited both by CrmA/SPI-2 and by a peptide spanning the lamin A apoptotic cleavage site. These results reveal that CrmA/SPI-2 can inhibit a caspase responsible both for lamin A cleavage and for the nuclear disintegration characteristic of apoptosis (Takahashi, 1996).

To investigate the involvement of different proteases in the execution step of apoptosis and to determine their intracellular location, isolated rat thymocyte nuclei were incubated either in the presence of Ca2+ and Mg2+ or with cytosolic extract from Jurkat T lymphocytes treated with anti-Fas (APO-1, CD-95) antibody. Inhibitors of caspases, VADcmk and DEVDcho, are not effective in hindering Ca2+-induced apoptotic changes in isolated nuclei, but do prevent similar changes in nuclei treated with the cytosolic extract from apoptotic Jurkat cells. In contrast, the inhibitor of the Ca2+-regulated, nuclear scaffold-associated serine protease, AAPFcmk, is able to inhibit lamin B1 breakdown, as well as chromatin cleavage in nuclei incubated in the presence of Ca2+ and Mg2+, but it only partially prevents the same changes induced with cytosolic extract. These findings provide evidence for the involvement of at least two proteases in lamin cleavage. One belongs to the caspase family: to cleave lamins this enzyme must be translocated from the cytoplasm into the nucleus. The second protease has a nuclear location and is activated by Ca2+. Neither of these two lamin-cleaving proteases is responsible for the cleavage of another nuclear target protein, poly(ADP-ribose)polymerase (PARP), during apoptosis (Zhivotovsky, 1997).

The fate of the nuclear envelope (NE) has been studied in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), a selective and conserved cleavage of LB, LAP2 and Nup153 is observed. In lymphoid cells, the rate of cleavage of these markers is independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 is probably caspase 3, since (1) cleavage of both proteins is specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. Since LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, it is suggested that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study (Buendia, 1999).

A-type lamins form distinct filamentous networks with differential nuclear pore complex associations

The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10-30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform. Two A-type lamin isoforms, lamins A and C (LaA and LaC; see Drosophila Lamin), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, BioID, an in vivo proximity-labeling method was used to identify differential interactors of A-type lamins. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, this Tpr association was demonstrated to be mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed construction of a nanometer-scale model of the mammalian nuclear lamina. These data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution (Xie, 2016).

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Lamin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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