Biochemical, genetic, and functional analyses of the phosphorylation sites on the Epstein-Barr virus-encoded oncogenic latent membrane protein LMP-1.

LMP-1 is the only Epstein-Barr virus-encoded latent protein known to have the properties of a transforming oncogene in rodent fibroblasts and the only latent protein, besides EBNA-1, detected in nasopharyngeal carcinoma and Hodgkin's lymphoma biopsies. LMP-1 is characterized by serine/threonine phosphorylation and rapid turnover (half-life, 2 to 3 h) due to specific proteolytic cleavage, which causes release of a phosphorylated C-terminal fragment (p25) into the cytoplasm. We used biochemical, functional, and mutational analyses to identify sites of phosphorylation. All of the phosphorylation sites detected lie in the C-terminal domain. In particular, we identified S-313 and T-324 as functionally important sites. Prevention of phosphorylation at S-313, by altering it to a glycine, prevented detectable phosphorylation of both LMP-1 and p25, indicating that it is a major site on both forms of the molecule. However, lack of detectable phosphorylation had no effect on p25 cleavage or on the ability of LMP-1 to transform Rat-1 fibroblasts. Alteration of S-313 to an aspartate resulted in a form of LMP-1 that was toxic to Rat-1 cells. Alteration of T-324 to a glycine residue had no detectable effect on the ability of LMP-1 to become serine phosphorylated or transform Rat-1 cells. Alteration of T-324 to a glutamate, however, inhibited all detectable phosphorylation and resulted in a form of LMP-1 that was unable to transform Rat-1 fibroblasts. These results are discussed in the context of a model in which LMP-1 function is modulated by phosphorylation and dephosphorylation at S-313 and T-324.     

The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.     

Sorting signals within the Saccharomyces cerevisiae sporulation-specific dityrosine transporter, Dtr1p, C terminus promote Golgi-to-prospore membrane transport

During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.     

Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis

The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-kappaB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1's lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1's polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1's growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1's signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1's lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-kappaB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1's first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1's polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-kappaB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activity's being linked to individual membrane-spanning domains within LMP-1's polytopic transmembrane domain.     

An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.     

Structural and functional separation of the N- and C-terminal domains of the yeast V-ATPase subunit H

The H subunit of the yeast V-ATPase is an extended structure with two relatively independent domains, an N-terminal domain consisting of amino acids 1-348 and a C-terminal domain consisting of amino acids 352-478. We have expressed these two domains independently and together in a yeast strain lacking the H subunit (vma13Delta mutant). The N-terminal domain partially complements the growth defects of the mutant and supports approximately 25% of the wild-type Mg(2+)-dependent ATPase activity in isolated vacuolar vesicles, but surprisingly, this activity is both largely concanamycin-insensitive and uncoupled from proton transport. The C-terminal domain does not complement the growth defects, and supports no ATP hydrolysis or proton transport, even though it is recruited to the vacuolar membrane. Expression of both domains in a vma13Delta strain gives better complementation than either fragment alone and results in higher concanamycin-sensitive ATPase activity and ATP-driven proton pumping than the N-terminal domain alone. Thus, the two domains make complementary contributions to structural and functional coupling of the peripheral V(1) and membrane V(o) sectors of the V-ATPase, but this coupling does not require that they be joined covalently. The N-terminal domain alone is sufficient for activation of ATP hydrolysis in V(1), but the C-terminal domain is essential for proper communication between the V(1) and V(o) sectors.     

Chimeric proteins suggest that the catalytic and/or C-terminal domains give CesA1 and CesA3 access to their specific sites in the cellulose synthase of primary walls

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3(WT) protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3(rsw5)) does not.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

P1 ParB Domain Structure Includes Two Independent Multimerization Domains

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.     

The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization

Interactor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the first plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green fluorescent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1DeltaN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1DeltaN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1DeltaN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.     

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.     

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.     

Characterization of an immortalizing N-terminal domain of polyomavirus large T antigen.

Polyomavirus large T antigen has an N-terminal domain of approximately 260 amino acids which can immortalize primary cells but lacks sequences known to be required for DNA binding and replication. Treatment of full-length large T with either V8 protease or chymotrypsin yields an N-terminal fragment of 36 to 40 kDa and a C-terminal fragment of approximately 60 kDa. This finding suggests a division of the protein into two domains. Proteolysis experiments show that the N-terminal domain does not have strong physical association with the rest of the protein. It also does not self-associate. A construct expressing only the N-terminal 259 amino acids is sufficient for immortalization. The independently expressed N-terminal domain is multiply phosphorylated, although at a lower level than the same region in full-length large T. The 259-residue protein binds to both pRb and p107 with somewhat lower efficiency than the full-length protein.     

Identification of conserved amino acids N-terminal of the PKC epsilon C1b domain crucial for protein kinase C epsilon-mediated induction of neurite outgrowth

We have shown previously that protein kinase C (PKC) epsilon can induce neurite outgrowth independently of its catalytic activity via a region encompassing its C1 domains. In this study we aimed at identifying specific amino acids in this region crucial for induction of neurite outgrowth. Deletion studies demonstrated that only 4 amino acids N-terminal and 20 residues C-terminal of the C1 domains are necessary for neurite induction. The corresponding regions from all other novel isoforms but not from PKCalpha were also neuritogenic. Further mutation studies indicated that amino acids immediately N-terminal of the C1a domain are important for plasma membrane localization and thereby for neurite induction. Addition of phorbol ester made this construct neurite-inducing. However, mutation of amino acids flanking the C1b domain reduced the neurite-inducing capacity even in the presence of phorbol esters. Sequence alignment highlighted an 8-amino acid-long sequence N-terminal of the C1b domain that is conserved in all novel PKC isoforms. Specifically, we found that mutations of either Phe-237, Val-239, or Met-241 in PKCepsilon completely abolished the neurite-inducing capacity of PKCepsilon C1 domains. Phorbol ester treatment could not restore neurite induction but led to a plasma membrane translocation. Furthermore, if 12 amino acids were included N-terminal of the C1b domain, the C1a domain was dispensable for neurite induction. In conclusion, we have identified a highly conserved sequence N-terminal of the C1b domain that is crucial for neurite induction by PKCepsilon, indicating that this motif may be critical for some morphological effects of PKC.