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[Background] Cognitive impairment is one of the most disabling nonmotor symptoms in Parkinson's disease (PD). Recently, a genome‐wide association study in Alzheimer's disease has identified the PICALM rs3851179 polymorphism as one of the most significant susceptibility genes for Alzheimer's disease after APOE. The aim of this study was to determine the potential role of PICALM and its genetic interaction with APOE in the development of cognitive decline in PD., [Methods] A discovery cohort of 712 patients with PD were genotyped for PICALM (rs3851179) and APOE (rs429358 and rs7412) polymorphisms. The association of PICALM and APOE–PICALM genetic interaction with cognitive dysfunction in PD was studied using logistic regression models, and the relationship of PICALM with cognitive decline onset was assessed with Cox regression analysis. PICALM effect was then replicated in an international, independent cohort (Parkinson's Progression Markers Initiative, N = 231)., [Results] PICALM rs3851179 TT genotype was significantly associated with a decreased risk of cognitive impairment in PD (TT vs. CC + CT, P = 0.041, odds ratio = 0.309). Replication studies further demonstrated its protective effect on cognitive impairment in PD. In addition, the protective effect of the PICALM rs3851179 TT genotype was more pronounced in the APOE ε4 (−) carriers from the discovery cohort (P = 0.037, odds ratio = 0.241), although these results were not replicated in the Parkinson's Progression Markers Initiative cohort., [Conclusions] Our results support the fact that PICALM is associated with cognitive impairment in PD. The understanding of its contribution to cognitive decline in PD could provide new targets for the development of novel therapies. © 2020 International Parkinson and Movement Disorder Society., Research funding: Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía. Grant Numbers: CTS‐7685, CVI‐02526; Consejería de Salud, Junta de Andalucía. Grant Numbers: C‐0048‐2017, PE‐0186‐2019, PE‐0210‐2018, PI‐0459‐2018, PI‐0471‐2013; Fundación Alicia Koplowitz; Instituto de Salud Carlos III. Grant Numbers: B‐0007‐2019, CM18/00142, MSII14/00018, PI14/01823, PI16/01575, PI18/01898, PI19/01576; Ministerio de Educación, Cultura y Deporte. Grant Number: FPU16/05061.

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here, we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved coat‐associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high‐resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below the lattice. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the coat. We propose that this design enables an efficient and regulated function of the endocytic coat during endocytic vesicle formation., FRET‐based protein proximity mapping reveals the nanoscale organization of the conserved endocytic coat: Coat proteins form several functional layers above and below the clathrin lattice with key endocytic factors transversing through it.

How clathrin-mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin-coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1-43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co-immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.

The CALM–AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

The ZNF198-fibroblast growth factor receptor-1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198-FGFR1 have been identified. Using a genetically engineered HEK 293 cell system, we have identified proteins that are specifically phosphorylated in the presence of the fusion kinase using anti-phosphotyrosine immunoprecipitation and MS. Compared with 293 cells expressing exongenous wild type FGFR1, ZNF198-FGFR1 is associated with phosphorylation of several proteins including SSBP2, ABL, FLJ14235, CALM and TRIM4 proteins. The specificity of the phosphorylation events in the SSBP2 and ABL proteins, which have previously been implicated in leukemogenesis, was further confirmed independently using immunoprecipitation with protein-specific antibodies and Western blotting. The MS analysis also identified the phosphorylation events in the ZNF198 moiety in the chimeric protein that might be related to its function. These studies identify the intersection of several different leukemia-related pathways in the development of this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions.

An interstitial deletion in the middle and distal part of chromosome 14 is a rare chromosomal abnormality characterized by a wide spectrum of phenotypic manifestations. We present a patient with a nearly 20 Mb interstitial deletion of chromosome 14q24.3q32.13 determined by FISH, that is associated with minor dysmorphic features, developmental delay, absent speech and auditory neuropathy. The deleted region contains 130 known genes, among them 48 with reported function or association with human disease. The patient's phenotype is compared with interstitial deletions of the distal part of chromosome 14 reported previously. We hypothesize, that there is (are) a gene (genes) in the 14q32.11-q32.13 that is (are) important for the hearing process and for which haploinsufficiency can cause auditory neuropathy. Several genes in the region, among them calmodulin, chromogranin A, the goosecoid and FOXN3, can contribute to the observed phenotype. Detailed mapping in additional patients with 14q32 deletions and hearing loss could further define the candidate region.

The t(9;22)(q34;q11) translocation occurs in chronic myeloid leukemia (CML) and adult B-cell acute lymphoblastic leukemia (ALL), leading to fusion of BCR to ABL1 and constitutive activation of ABL1 tyrosine kinase activity. The main BCR–ABL1 breakpoints result in P190 BCR–ABL1 or P210 BCR–ABL1 fusion proteins. The latter is found in almost all cases of CML and in one third of the cases of t(9;22)-positive adult B-ALL. P190 BCR–ABL1 is found in the remaining two thirds of t(9;22)-positive adult B-ALL cases but only exceptionally in CML. We describe here the first case of t(9;22)(q34;q11) associated with t(10;11)(p13;q14) in acute monocytic leukemia. The recurrent t(10;11)(p13;q14) translocation, usually found in acute myeloid leukemia (AML) and T-ALL, merges PICALM to MLLT10. RT-PCR enabled identification of PICALM–MLLT10 and BCR–ABL1 e1–a2 fusion transcripts; in the context of chronic and acute myeloid leukemia, the latter usually has a monocytic presentation. We also identified overexpression of HOXA9, a gene essential to myeloid differentiation that is expressed in PICALM–MLLT10 and MLL-rearranged acute leukemias. This case fits with and extends a recently proposed multistage AML model in which constitutive activation of tyrosine kinases by mutations (BCR–ABL1) are associated with deregulation of transcription factors central to myeloid differentiation (HOXA9 secondary to PICALM–MLLT10). © 2006 Wiley-Liss, Inc.

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the α-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.

Clathrin-coated vesicles mediate a variety of endocytosis pathways in cells, including endocytic events at synapses. AP180 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are clathrin accessory proteins that promote the formation of clathrin-coated vesicles. Both proteins bind to membrane lipids through their epsin N-terminal homology domains and interact with clathrin and related protein components through their carboxyl-terminal peptide motifs. We examine their neuronal expression and synaptic distribution. We show that both proteins are detected in synapses but demonstrate different distribution patterns. AP180 is located predominantly in presynaptic profiles, whereas CALM is found nonselectively in pre- and postsynaptic profiles and also in perisynaptic processes. These observations reveal an unexpected relationship between AP180 and the presumed non-neuronal homologue CALM. We propose that both AP180 and CALM function as endocytic accessory proteins at synapses, but each may regulate distinct clathrin pathways. J. Comp. Neurol. 481:58–69, 2005. Published 2004 Wiley-Liss, Inc.

Rearrangements involving the MLL gene at chromosome band 11q23 are common in infant acute myeloid leukemias (AMLs). We recently encountered an infant patient with rapidly progressive AML whose leukemic cells harbored a previously undescribed MLL rearrangement involving an inversion of 11q [inv(11)(q14q23)]. We used panhandle PCR to determine that this rearrangement juxtaposed the MLL (Mixed-Lineage Leukemia) gene to the CALM (Clathrin Assembly Lymphoid Myeloid leukemia) gene at 11q14 – q21. The CALM protein participates in recruitment of clathrin to internal membrane surfaces, thereby regulating vesicle formation in both endocytosis and intracellular protein transport. Intriguingly, CALM has been identified in other cases of AML as a translocation partner for the AF10 gene, which has independently been found to be an MLL partner in AML. We identified the MLL-CALM fusion transcript (but not the reciprocal CALM-MLL transcript) in leukemia cell RNA by RT-PCR. The predicted 1803 amino acid MLL-CALM fusion protein includes amino-terminal MLL domains involved in transcriptional repression, and carboxy-terminal CALM-derived clathrin-binding domains. The genomic breakpoint in MLL is in the 7th intron (within the breakpoint cluster region); the corresponding CALM breakpoint is in the 7th CALM intron. In contrast, breakpoints in CALM-AF10 translocations lie in the 17th–19th CALM introns (30 kb downstream); also, in these translocations, CALM provides the 5 end of the fusion transcript. Together with its previously recognized association with AF10 in AML, the identification of CALM as an MLL fusion partner suggests that interference with clathrin-mediated trafficking pathways may be an underappreciated mechanism in leukemogenesis. © 2003 Wiley-Liss, Inc.

Background Evidence has emerged indicating that the e4 allele of APOE and PICALM interact in conferring risk of Alzheimer's disease (AD). The biologic basis of this interaction is unclear, but it is likely to have phenotypic relevance and contribute to the structural and clinical heterogeneity of AD. Methods The aim of this study was to investigate interaction effects of the APOE e4 allele and the alleles at the single-nucleotide polymorphism rs3851179 located in the PICALM locus. We analyzed brain volumes and cognitive phenotypes of 165 patients with early AD dementia. Results There was a synergistic adverse effect of homozygosity for the PICALM risk allele G in rs3851179 and APOE e4 on volume in prefrontal and performance on the Trail Making Test A, which is sensitive to processing speed and working memory function. Conclusions The data suggest a neural mechanism for APOE–PICALM interactions in patients with manifest AD and indicate that the PICALM genotype modulates both brain atrophy and cognitive performance in APOE e4 carriers.

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.

The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes. The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system. AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity. In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins. To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed. Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function. Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells. These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast. Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.

Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP-1 and AP-2 are the most extensively characterized. The function of the recently described adaptor complex AP-3, which is similar to AP-1 and AP-2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP-3 is able to interact with the tail of the lysosomal integral membrane protein LIMP-II and that this binding depends on a DEXXXLI sequence in the LIMP-II tail. Furthermore, AP-3 bound to the cytoplasmic tail of the melanosome-associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP-II and tyrosinase either did not interact, or only interacted poorly, with AP-1 or AP-2. In contrast, the cytoplasmic tails of other membrane proteins containing di-leucine and/or tyrosine-based sorting signals did not bind AP-3, but AP-1 and/or AP-2. This points to a function of AP-3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di-leucine-based sorting motifs.

In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are required for viability. We describe here the molecular analysis of four mutations that eliminate the requirement for Yck activity. These mutations alter proteins that resemble the four subunits of clathrin adaptors (APs), with highest sequence similarity to those of the recently identified AP-3 complex. The four yeast subunits are associated in a high-molecular-weight complex. These proteins have no essential function and are not redundant for function with other yeast AP-related proteins. Combination of suppressor mutations with a clathrin heavy chain mutation (chc1-ts) confers no synthetic growth defects. However, a yck(ts) mutation shows a strong synthetic growth defect with chc1-ts. Moreover, endocytosis of Ste3p is dramatically decreased in yck(ts) cells and is partially restored by the AP suppressor mutations. These results suggest that vesicle trafficking at the plasma membrane requires the activity of Yck protein kinases, and that the new AP-related complex may participate in this process.

F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain. This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3. It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution. This may be important for the regulation of quanta1 size during neurotransmission. We also express the 33 kD NH2-terminus of F1-20/AP-3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH2-terminus of F1-20/AP-3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH2-terminus of F1-20/ AP-3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH2-terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated.

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.

The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal plasma membrane (Su et al., 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals (Perry et al., 1991). In this report we describe double-labelling experiments with avian embryonic striated muscle fibers demonstrating the exclusive presence of the brain-specific protein at the neuromuscular junction. We used indirect rhodamine immunofluorescence labeling with a monoclonal antibody (mAb-8G8) to mark the location of NP185 in muscle combined with fluorescein-alpha-bungarotoxin to mark the postsynaptic location of the acetylcholine receptors (AChRs). We show that the distribution of both NP185 and AChRs has an overall correlation, but the location of NP185 is circumscribed to presynaptic structures adjacent but not overlapping with postsynaptic structures displaying the AchRs. To confirm the identity of NP185, the molecule was extracted from both tissues, partially purified, immunoprecipitated, and identified in Western blots with the mAb 8G8. The mAb reacted with an identical 185 kD protein band purified from both tissues. Based on its properties and specific neuronal location, the NP185 molecule may function in motor nerve terminals by screening membrane proteins, identifying areas of the synaptic plasma membrane, and to anchor these elements with structural proteins for their recycling and transport within the neuronal cellular compartments.

We have established by peptide mapping and immunochemical analysis of purified clathrin assembly protein preparations from bovine brain, that the cluster of components of mol. wt 100-120 kd fall into four classes, which we term alpha, beta, beta' and gamma. The beta and beta' proteins are immunologically related and generate a series of common tryptic peptides. The same criteria reveal no such homologies between the alpha, beta(beta') and gamma polypeptides. The so-called HA-II assembly protein group contains equimolar amounts of alpha and beta class polypeptides, which are shown to interact with each other. In the HA-I group assembly protein complex gamma and beta' class polypeptides form a stoichiometric complex. Immunofluorescence microscopy reveals that the HA-I complex is specifically associated with clathrin-coated membranes in the Golgi region of cultured cells, whereas the HA-II complex appears to be restricted to coated pits on the plasma membrane. The data lead to the tentative conclusion that the clathrin assembly proteins are involved in the recognition of the intracellular targets by uncoated vesicles.

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.