Acetylcholine receptor assembly is stimulated by phosphorylation of its gamma subunit

Different combinations of Torpedo acetylcholine receptor (AChR) subunits stably expressed in mouse fibroblasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes (alpha 2 beta gamma delta) were labeled with 32P, only gamma and delta subunits were phosphorylated. Forskolin, which causes a 2- to 3-fold increase in AChR expression by stimulating subunit assembly, increased unassembled gamma phosphorylation, but had little effect on unassembled delta. The forskolin effect on subunit phosphorylation was rapid, significantly preceding its effect on expression. The pivotal role of the gamma subunit was established by treating alpha beta gamma and alpha beta delta cell lines with forskolin and observing increased expression of only alpha beta gamma complexes. This effect was also observed in alpha gamma, but not alpha delta cells. We conclude that the cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of unassembled gamma subunits, which leads to increased efficiency of assembly of all four subunits.     

Adjacent basic amino acid residues recognized by the COP I complex and ubiquitination govern endoplasmic reticulum to cell surface trafficking of the nicotinic acetylcholine receptor alpha-Subunit

The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of alpha-gamma-alpha-delta-beta. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg(313)-Lys(314) sequence in the large cytoplasmic loop of the alpha-subunit to K314Q promotes the trafficking of the mutant unassembled alpha-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q alpha-subunit with gamma-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type alpha-subunits when co-expressed with the beta-, delta-, and gamma-subunits. This suggests that the Arg(313)-Lys(314) sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the alpha-subunit. Although unassembled K314Q alpha-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q alpha-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface.     

Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit

We investigated whether the assembly/disassembly of the 26S proteasome is regulated by phosphorylation/dephosphorylation. The regulatory complex disassembled from the 26S proteasome was capable of phosphorylating the p45/Sug1/Rpt6 subunit, suggesting that the protein kinase is activated upon dissociation of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regulatory complex was found to be incorporated into the 26S proteasome. When the 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 subunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. These results indicate that the 26S proteasome is disassembled into the constituent subcomplexes by dephosphorylation and that it is assembled by phosphorylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly associated with the 20S proteasome alpha-subunit C3 in a phosphorylation-dependent manner.     

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25–28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.     

Distinct phosphoisoforms of the Xenopus Mcm4 protein regulate the function of the Mcm complex

Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.     

Formation of the alpha-bungarotoxin binding site and assembly of the nicotinic acetylcholine receptor subunits occur in the endoplasmic reticulum

During the process by which newly synthesized subunits of the nicotinic acetylcholine receptor (stoichiometry = alpha 2 beta gamma delta) mature and acquire the properties of the fully functional cell surface receptor, they undergo numerous covalent and noncovalent modifications. Using ligand-mediated and subunit-specific immunoprecipitation, four forms in the maturation of the alpha subunit can be detected: the primary translation product; alpha subunit that can bind alpha-bungarotoxin; alpha subunit assembled with the other subunits; and surface receptor. The alpha subunit acquires the ability to bind alpha-bungarotoxin with a t1/2 of approximately 40 min after translation and becomes assembled with a t1/2 of 80 min after translation. Using metabolic labeling and sucrose gradient fractionation, we have determined the subcellular location of alpha subunit when it acquires the ability to bind alpha-bungarotoxin and when it is assembled. Golgi membranes were identified across the gradient by the enzymatic activities UDP-galactose:N-acetylglucosamine galactosyltransferase and alpha-mannosidase. Endoplasmic reticulum membranes were identified by the enzymatic activity glucose-6-phosphatase and by the presence of newly synthesized alpha and beta subunits. Pulse-labeled alpha subunit that bound alpha-bungarotoxin was first detected co-migrating in the gradient with the glucose-6-phosphatase activity. Therefore, the capacity to bind alpha-bungarotoxin was acquired while the alpha subunit was in the endoplasmic reticulum. Assembled alpha subunit was detected by immunoprecipitating with an anti-beta subunit-specific monoclonal antibody. By this method, assembled receptor was first detected 15 min after translation in both the endoplasmic and Golgi portions of the gradient. To validate this method of detecting assembled receptor, we determined the sedimentation coefficient of the receptor subunits in the endoplasmic reticulum. Both unassembled subunits with sedimentation coefficients of 5 S and assembled receptor with a sedimentation coefficient of 9 S were recovered from the endoplasmic reticulum portion of the gradient. Thus, our data concerning the subcellular site of assembly are consistent with assembly occurring in the endoplasmic reticulum followed by rapid transport to the Golgi.     

Regulation of nicotinic receptor expression by the ubiquitin-proteasome system

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.     

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.     

BIP associates with newly synthesized subunits of the mouse muscle nicotinic receptor

A slow conformational change in newly synthesized acetylcholine receptor subunits is thought to be a requisite step in the biogenesis of this multi-subunit transmembrane glycoprotein. Previously, we demonstrated that this early conformational change within the alpha-subunit was inefficient and dependent upon disulfide bond formation (Blount, P. and J.P. Merlie. 1990. J. Cell Biol. 111:2613-2622). Here we show that newly synthesized acetylcholine receptor subunits and subunit complexes in the muscle-like cell line, BC3H-1, are associated with Bip, a ubiquitous binding protein of the endoplasmic reticulum. Characterization of the Bip/alpha-subunit complex in stably transfected fibroblasts revealed that Bip associates with newly synthesized unassembled alpha-subunit and some alpha gamma and alpha delta subunit complexes. Significantly, Bip does not associate well with the more mature form of the alpha-subunit containing an intramolecular disulfide bridge. Hence, Bip may play an important role in the conformational maturation and/or editing of unassembled AChR subunits and subunit complexes in vivo.     

The mitotic phosphorylation cycle of the cis-Golgi matrix protein GM130

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Balpha regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.     

Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro

The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid. The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit. In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head. Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization. Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS. In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.     

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

BiP forms stable complexes with unassembled subunits of the acetylcholine receptor in transfected COS cells and in C2 muscle cells

We have investigated the role of the immunoglobulin-binding protein (BiP) in the folding and assembly of subunits of the acetylcholine receptor (AChR) in COS cells and in C2 muscle cells. Immunoprecipitation in COS cells showed that alpha, beta, and delta subunits are associated with BiP. In the case of the alpha subunit, which first folds to acquire toxin-binding activity and is then assembled with the other subunits to form the AChR, BiP was associated only with a form that is unassembled and does not bind alpha-bungarotoxin. Similar results were found in C2 cells. Although the alpha and beta subunits of the AChR are minor membrane proteins in C2 cells, they were prominent among the proteins immunoprecipitated by antibodies to BiP, suggesting that BiP could play a role in their maturation or folding. In pulse-chase experiments in C2 cells, however, labeled alpha subunit formed a stable complex with BiP that was first detected after most of the alpha subunit had acquired toxin-binding activity and whose amount continued to increase for several hours. These kinetics are not compatible with a role for the BiP complex in the folding or assembly pathway of the AChR, and suggest that BiP is associated with a misfolded form of the subunit that is slowly degraded.     

Rapsyn‐mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high‐density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the α subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn‐induced clustering of the AChR β, γ, and δ subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into αδ dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild‐type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn‐mediated clustering of an assembly intermediate, the αδ dimer. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 486–501, 2003     

Lamin B methylation and assembly into the nuclear envelope

Lamin B is reversibly methyl-esterified and phosphorylated during the mammalian cell cycle. In order to study the role of methylation in lamin B function, we isolated mitotic cells in the presence of the microtubule inhibitor, nocodazole. Following removal of nocodazole, methylation of mitotic lamin B was found to precede its assembly into the nuclear envelope as cells exited mitosis. Very little additional methylation took place on assembled lamins. We were able to slow the rate of lamin B methylation with methylthioadenosine (MTA). A delay in lamin B methylation was accompanied by a corresponding delay in assembly of lamin B into the envelope. The delay was approximately 20-30 min beyond the typical 60-70 min usually required. Assembly of lamins A and C, which are not methylated, was also delayed by MTA, although to a lesser degree, suggesting that an interaction between the lamins is necessary for formation of the nuclear envelope. Chromatin decondensation was also slowed in the presence of MTA. Other inhibitors of methylation which had no inhibitory effect on the methyl esterification of lamin B were tested and found to have no effect on envelope assembly or chromatin decondensation. These results were obtained with Chinese hamster ovary cells as well as with the stem cell line, PC 13. Dephosphorylation of lamin B normally follows a time course similar to that of nuclear envelope assembly. In the presence of MTA, however, lamin B assembly was slowed with little effect on dephosphorylation. This resulted in a large population of dephosphorylated, but unassembled, lamin B protein, demonstrating that dephosphorylation is not sufficient for envelope assembly. The lack of effect on the time course of dephosphorylation also suggests that MTA is not acting upstream of the methylation event.     

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.     

Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering

At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR beta subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the beta subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the beta subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus beta subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type beta subunit but not tyrosine-minus beta subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus beta subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR beta subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.     

Phycobilisome linker proteins are phosphorylated in Synechocystis sp. PCC 6803

The controversial issue of protein phosphorylation from the photosynthetic apparatus of Synechocystis sp. PCC 6803 has been reinvestigated using new detection tools that include various immunological and in vivo labeling approaches. The set of phosphoproteins detected with these methods includes ferredoxin-NADPH reductase and the linker proteins of the phycobilisome antenna. Using mutants that lack a specific set of linker proteins and are affected in phycobilisome assembly, we show that the phosphoproteins from the phycobilisomes correspond to the membrane, rod, and rod-core linkers. These proteins are in a phosphorylated state within the assembled phycobilisomes. Their dephosphorylation requires partial disassembly of the phycobilisomes and further contributes to their complete disassembly in vitro. In vivo we observed linker dephosphorylation upon long-term exposure to higher light intensities and under nitrogen limitation, two conditions that lead to remodeling and turnover of phycobilisomes. We conclude that this phosphorylation process is instrumental in the regulation of assembly/disassembly of phycobilisomes and should participate in signaling for their proteolytic cleavage and degradation.     

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.