(Received for publication, February 3, 1997, and in revised form, March 5, 1997)
From the Departments of Neuroscience and Physiology, The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205 and
the Departments of Surgery and Physiology, University of
Maryland School of Medicine, Baltimore, Maryland 21201
Although the integral membrane proteins that
catalyze steps in the biosynthesis of neuroendocrine peptides are known
to contain routing information in their cytosolic domains, the proteins
recognizing this routing information are not known. Using the yeast
two-hybrid system, we previously identified P-CIP10 as a protein
interacting with the cytosolic routing determinants of peptidylglycine
-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic
protein with nine spectrin-like repeats and adjacent Dbl homology and
pleckstrin homology domains typical of GDP/GTP exchange factors. In the
adult rat, expression of P-CIP10 is most prevalent in the brain.
Corticotrope tumor cells stably expressing P-CIP10 and PAM produce
longer and more highly branched neuritic processes than nontransfected
cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to
selected members of the Rho subfamily of small GTP binding proteins
(Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl
family of proteins, may serve as part of a signal transduction system
linking the catalytic domains of PAM in the lumen of the secretory
pathway to cytosolic factors regulating the cytoskeleton and signal
transduction pathways.
Cytosolic proteins are involved in the formation of secretory
granules (1-6) and in the trafficking and localization of integral membrane proteins needed for the synthesis of bioactive peptides (7-16). We have used one of the few integral membrane proteins known
to be involved in the biosynthesis of neuropeptides, peptidylglycine -amidating monooxygenase (PAM),1 to
search for cytosolic proteins involved in these processes (17). PAM
is a bifunctional enzyme and integral membrane forms contain an
NH2-terminal signal sequence, the two catalytic domains that catalyze the sequential reactions required for peptide amidation, a single transmembrane domain, and a short cytosolic domain (18).
PAM is involved in the production of all -amidated peptides and
functions only after neuroendocrine-specific endoproteases and
carboxypeptidases have exposed the COOH-terminal glycine residue that
serves as the nitrogen donor for amide formation (19). Immunocytochemical evidence indicates that PAM begins to function in
the trans-Golgi network (TGN), but most peptide amidation
occurs in immature secretory granules (20). Using immunoelectron
microscopy, integral membrane forms of PAM have been localized to the
TGN, especially to distal tubuloreticular regions, and to large dense core vesicles (21).
When expressed independently in neuroendocrine cells, each lumenal
catalytic domain of PAM is targeted to large dense core vesicles (22).
Integral membrane forms of PAM are localized to the TGN region of both
neuroendocrine and nonneuroendocrine cells (7, 8). A small percentage
of membrane PAM is present on the cell surface or in endosomes at
steady state. Elimination of the distal 40 amino acids of the 86-amino
acid cytosolic domain results in relocation of membrane PAM to the
plasma membrane (8, 9). When transferred to a plasma membrane protein
such as the interleukin 2 receptor -chain (Tac), the cytosolic
domain of PAM directs the majority of the protein to the TGN region and confers the ability to undergo internalization from the plasma membrane
(9).
Mutation of a tyrosine residue in the COOH-terminal domain of PAM greatly diminishes internalization of PAM from the cell surface without dramatically altering its TGN localization (9). The TGN localization of membrane PAM is greatly compromised upon deletion of an 18-amino acid domain that includes the tyrosine residue essential for internalization. Integral membrane PAM proteins are phosphorylated, and mutagenesis studies indicate that phosphorylation affects routing (23).
Using a rat hippocampal library and the yeast two-hybrid system, we recently identified partial cDNAs encoding two PAM COOH-terminal interactor proteins (P-CIPs) (17). The biological relevance of these interactions is supported by the fact that the interactions are eliminated when the 18-amino acid segment identified as essential for proper routing of PAM is eliminated. P-CIP2 is similar to serine/threonine dual specificity protein kinases, while P-CIP10 contains at least five spectrin-like repeats (17). In this study we identify the full-length P-CIP10 protein as a member of the Dbl family of GDP/GTP exchange factors (24) and establish the phenotype of stably transfected AtT-20 cell lines expressing PAM-1 and P-CIP10.
The 2.0-kb P-CIP10
cDNA fragment identified using the yeast two-hybrid system (17) was
used to screen 1 × 106 plaque-forming units from a
random primed rat hippocampal cDNA library in -ZAPII
(Stratagene). Seven positive clones were plaque-purified, and the two
largest cDNA inserts recovered were overlapping 4.7-kb (clone
10/28) and 4.1-kb (clone 10/34) fragments. Both strands of these
cDNAs were sequenced completely; only the 5
ends of clones 10/28
(upstream sequence from nucleotide 36) and 10/34 (upstream sequence
from nucleotide 419) differed. Attempts to extend these sequences by
5
-rapid amplification of cDNA ends (RACE) were unsuccessful. The
five shorter cDNAs were fragments of the larger pieces. DNA
manipulations were carried out according to standard protocols.
Since no in-frame stop codon was identified in clones 10/28 and 10/34,
3 RACE was used to extend the 3
-end of the 10/28 cDNA (25).
Briefly, poly (A)+ RNA from adult rat parietal cortex (200 ng) or olfactory bulb (140 ng) was reverse transcribed with the Promega
reverse transcription system (Madison, WI) using a RACE hybrid primer
(RHP) 5
-GGAATTCGAGCTCATCGAT17-3
(0.75 µM) and avian myeloblastosis virus reverse transcriptase (15 units). After the initial 35-cycle amplification using
5
-CAGGATGCCTTTCAAGTG-3
(nt 4375-4392 of final P-CIP10a) and RHP, an
aliquot of the PCR product was used in a nested PCR with
5
-CCTCTAGAGCACCCCATCCTCAGACAAT-3
(nt 4592-4611 of
P-CIP10a) and RHP. A 770-base pair fragment (674 nt of new sequence)
from independent amplifications of both tissues was purified, subcloned
into pBluescript II (SK
) (pBS), and sequenced. The additional
3
-sequence contained no in-frame stop codon, so 3
-RACE was repeated
as above using sense primers 5
-CTGCTTCTTCCCCCTGGTGA-3
(nt 5097-5116
of P-CIP10a) for the first round of amplification and
5
-GGTCTAGAATGGAGGCAAGTCTGAGT-3
(nt 5144-5164 of
P-CIP10a) for the second round of PCR reactions with the same RHP. The
650-base pair fragment (444 nt of new 3
sequence) obtained in this
second 3
-RACE reaction had an in-frame stop codon. Reverse
transcriptase-PCR was used to verify that the novel sequence contained
in the 3
-RACE products was contiguous to clone 10/28.
Full-length P-CIP10a (10/28 cDNA at the 5 end) and P-CIP10b (10/34
cDNA at the 5
end) cDNAs were constructed using a
PCR-generated pBS.P-CIP10-3
(nt 4255-5724) intermediate. Full-length
P-CIP10a cDNA (pBS.P-CIP10a) was created by a three-way ligation of
Bsp106I/BamHI-digested pBS, a 4.45-kb fragment
obtained from clone 10/28 by digestion with Bsp106I
(5
-MCS)-AspEI (nt 4401) and a 1.32-kb fragment obtained from AspEI/BamHI-cut pBS.P-CIP10-3
. To create
the full-length P-CIP10b cDNA (pBS.P-CIP10b), clone 10/34 was
digested with BstBI (nt 2674) and XbaI (3
-MCS),
and the 10/34 sequence downstream of BstBI was replaced with
the 3.4-kb BstBI (nt 2291 of P-CIP10a)-XbaI (3
-MCS) fragment from pBS.P-CIP10a.
Construction of pGEX-CIP10, an expression vector encoding a GST fusion protein containing P-CIP10a (aa 447-1138), was described (17). To construct pET-HisDH, a bacterial expression vector encoding all of the Dbl homology (DH) domain and most of the pleckstrin homology (PH) domain of P-CIP10a (P-CIP10a (aa 1254-1537)), the 857-base pair fragment from clone 10/28 was subcloned into pET28a (Novagen) in-frame with the histidine tag. A mammalian expression vector encoding full-length P-CIP10a (pSCEP.P-CIP10a) was constructed by inserting the full-length cDNA piece from pBS.P-CIP10a into pSCEP (26). To construct pBS.Myc.P-CIP10, the c-Myc epitope (underlined) with a Gly5 linker (MEQKLISEEDLNGGGGG) was joined in-frame to Gly5 of P-CIP10a using standard methods. The full-length cDNA insert was then transferred to pSCEP to generate p.SCEP.Myc.P-CIP10. All PCR-generated cDNA was confirmed by DNA sequencing.
In Vitro Transcription/Translation and Northern Blot AnalysisTruncated forms of P-CIP10 cDNAs were used as
templates for in vitro transcription and translation
reactions. pBS.10a (nt 1-1142) was generated by digesting pBS.P-CIP10a
with BglII (cuts at nt 1142) and BamHI (cuts in
3-MCS) and religating. pBS.10b (nt 1-1525) was generated in the same
manner from pBS.P-CIP10b. Radiolabeled proteins
([35S]methionine, 40 µCi/40-µl reaction; Amersham
Corp.) were synthesized using a rabbit reticulocyte lysate in
vitro transcription and translation system (TNT; Promega Corp.)
and analyzed by SDS-PAGE with or without prior immunoprecipitation.
Total RNA (10 µg) prepared from different adult rat tissues was
electrophoresed on 1% agarose gels containing formaldehyde (25).
Poly(A)+ RNA was prepared with the Promega
PolyATtract® mRNA isolation system II. RNA transferred
to nitrocellulose membranes was hybridized with a cDNA probe
encompassing nt 1363-3398 of P-CIP10a using standard protocols (25).
In situ hybridization was carried out as described (17).
Monoclonal antibody 6E6 recognizes PAM COOH-terminal cytosolic domain (CD) (9). Rabbit polyclonal antibody JH1764 was generated by immunization of adult female rabbits with purified recombinant rat PHM (aa 37-382) and recognizes native PHM (21). Rabbit antibody JH2007 was raised to a synthetic polypeptide (P-CIP10a (aa 992-1013)) linked to keyhole limpet hemocyanin with glutaraldehyde (Hazleton HRP, Inc., Denver, PA) (27). Rabbit polyclonal antibody JH2000 was raised by immunization with GST.P-CIP10a (aa 447-1138). The fusion protein purified by SDS-PAGE and electroelution was used as antigen. Antibody JH2000 recognizes full-length P-CIP10 only after denaturation. Monoclonal antibody 4C9 was generated to recombinant P-CIP10a (aa 1254-1537) (His-DH) expressed in Escherichia coli and purified by adsorption to nickel-bound His-Bind resin (Novagen) (9). Monoclonal antibody 9E10 to the Myc epitope (28) was prepared from conditioned medium or ascites fluid (Hazleton, HRP).
Cell Culture and Cell LinesAll cells were cultured in Dulbecco's modified Eagle medium/F-12 containing 10% fetal bovine serum (Hyclone, Logan, UT) and 10% Nu serum (Collaborative Research, Bedford, MA). Transfected cell lines were grown with appropriate drug selection: G418 (0.5 mg/ml) for pMt.Neo or pMc.Neo (Stratagene, La Jolla, CA) cotransfections; hygromycin (200 units/ml) for pCEP4 (Invitrogen) or pSCEP (26) cotransfections. AtT-20/PAM-1 cells were transfected with pSCEP.P-CIP10a or pSCEP.Myc.P-CIP10a using lipofection. Cells resistant to both hygromycin and G418 were selected, expanded, and screened for expression of P-CIP10 or Myc.P-CIP10 transcript by Northern blot analysis. Cell lines were subcloned and again screened by Northern blot analysis.
Biosynthetic Labeling, Subcellular Fractionation, and ImmunoprecipitationCells plated on 15-mm culture dishes and
grown to 70-90% confluency were incubated in methionine/cysteine-free
complete serum-free medium for 10 min and then labeled with the same
medium containing 1 mCi/ml [35S]methionine/cysteine
(ProMix; Amersham) for 15 or 30 min followed by a nonradioactive chase
in complete serum-free medium. Cells were either directly extracted
into SDS buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, 10 mM -mercaptoethanol) by incubation at 95 °C for 5 min
or were scraped into TMT buffer (10 mM sodium TES, pH 7.5, 20 mM mannitol, 1% Triton X-100) and subjected to three
cycles of freezing and thawing and centrifugation to pellet insoluble material. For the subcellular fractionation experiment, cells were
removed from the dishes by scraping into an isotonic buffer (50 mM HEPES-KOH, pH 7.5, 250 mM sucrose),
disrupted using a ball bearing cell cracker (15-µm clearance), and
subjected to differential centrifugation (23).
Immunoprecipitation of P-CIP10 utilized Ab JH2000 or Myc mAb 9E10 (23, 28). PAM-1 was immunoprecipitated using Ab JH1764 (22). Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by fluorography. Apparent molecular masses were determined using prestained molecular weight standards (Rainbow standards; Amersham). The capacities of Ab JH2000 and mAb 9E10 were determined by adding increasing amounts of AtT-20/P-CIP10a or AtT-20/Myc.P-CIP10 cell extract to a fixed amount of antibody plus radiolabeled in vitro translated Myc.P-CIP10.
Morphological Studies of Stably Transfected CellsLive AtT-20 cells were photographed at low magnification using phase contrast optics. Coded images were analyzed using a BioQuant TCW (version 3.00; R & M Biometrics, Nashville, TN) to record the total number of cells, number of cells with processes (longer than 1 cell body length), number of round cells, number of giant cells (cell body bigger than 3 nuclei), length of each process, and number of bifurcations (branch points). Four or five images were analyzed for each cell line (215-460 cells); after decoding, data were analyzed for statistical significance using a t test.
Complex Formation of GST-GTPases with P-CIP10E. coli transformed with vectors encoding GST-Rac1, GST-RhoA, and GST-Cdc42Hs were obtained from Dr. Richard C. Cerione (Cornell University, Ithaca, NY). The purified fusion proteins were prepared, dialyzed, bound to glutathione-Sepharose beads, and depleted of bound nucleotide (29, 30). AtT-20 cells expressing PAM-1 or PAM-1 with Myc.P-CIP10 were extracted into 20 mM Tris-HCl, pH 7.5, 50 mM NaCl containing 1% Triton X-100. After freezing and thawing three times, extracts were diluted with 3 volumes of the guanine-nucleotide depletion buffer (30) and centrifuged. For each binding reaction, about 50 µg of GST-GTPase bound to 50 µl of glutathione-Sepharose beads was mixed with an aliquot of cell extract containing 2 mg (0.25 mg/ml) of protein from nonlabeled cells or 5 × 107 cpm/ml acid-precipitable protein from radiolabeled cells. After mixing for 3 h at 4 °C, the beads were washed with nucleotide depletion buffer. The beads incubated with nonlabeled extracts were eluted with Laemmli buffer, and bound proteins were subjected to SDS-PAGE and Western blot analysis using Ab JH2000. The beads incubated with radiolabeled cell extracts were eluted by boiling in 50 mM Tris-HCl, pH 7.5, 1% SDS, diluted, and subjected to immunoprecipitation (9) with Ab JH2000.
P-CIP10 was
identified in a hippocampal/cortical cDNA library prepared from
3-week-old rat pups that had been subjected to a single maximal
electroconvulsive stimulus (17). Before trying to clone a full-length
P-CIP10 cDNA, we used Northern blot analysis to determine the size
of the P-CIP10 transcript and the tissues expressing the highest levels
of P-CIP10 (Fig. 1A). A somewhat heterogeneous set of P-CIP10 transcripts was visualized in total RNA
prepared from olfactory bulb, parietal cortex, and hippocampus, with
lower levels detected in hypothalamus and none detected in cerebellum.
P-CIP10 mRNA was detectable in total RNA prepared from kidney and
spleen but was not visualized in anterior or neurointermediate pituitary or atrium, tissues that contain high levels of PAM mRNA. Multiple forms of P-CIP10 mRNA were apparent in all of the tissues examined; when poly(A)+ mRNA from olfactory bulb and parietal cortex was subjected to Northern blot analysis, distinct bands of 8.0 and 5.7 kb were detected (Fig. 1A, inset).
A similar distribution of P-CIP10 transcripts was observed when in situ hybridization was performed on sections of adult rat brain (Fig. 1, B and D). P-CIP10 transcripts were prevalent in the olfactory bulb, including the internal granular layers, internal plexiform layer, mitral cell layer, and accessory olfactory bulb. P-CIP10 transcripts were also prevalent in all layers of the cerebral cortex, piriform cortex, and amygdala and in the dentate gyrus and CA1-3 regions of the hippocampus. P-CIP10 transcripts were present at lower levels in several hypothalamic structures, including the paraventricular, supraoptic, dorsomedial, and arcuate nuclei.
Isolation of Full-length P-CIP10 cDNAsOur partial
cDNA was substantially shorter than the P-CIP10 mRNAs observed
in tissues. We used the 2.0-kb P-CIP10 cDNA fragment to screen a
rat hippocampal cDNA library. The two largest cDNA fragments
isolated (10/28 and 10/34) were identical except at their 5-ends (Fig.
2A). No in-frame stop codons were found at either end of either cDNA. The GC content of the 5
-ends of both clones was high, and attempts to extend the sequences by 5
-RACE were
unsuccessful (25); sequence and in vitro translation data (see below) indicated that an initiator Met was included in each clone.
By sequentially employing 3
-RACE, we extended the sequence to include
an in-frame stop codon (Fig. 2A). No sequence diversity was
found in the newly amplified 3
-fragments. The fragments were assembled
to form two full-length P-CIP10 cDNAs, P-CIP10a (5
-end of 10/28)
and P-CIP10b (5
-end of 10/34) (Fig. 2A).
A single long open reading frame with a stop codon near the 3-end was
found in both P-CIP10 cDNAs. The GC-rich nature of the 5
-end of
both P-CIP10a and P-CIP10b and the presence of a single Met residue in
both unique regions raised the possibility that a transcriptional start
site was present in each clone. The nucleotide sequence surrounding
each Met agreed with the consensus translational initiation sequence
defined for higher eukaryotes (Fig. 2B) (31, 32). To
determine whether these potential translational initiation sites were
functional, we performed coupled in vitro transcription/translation reactions. We truncated P-CIP10a and P-CIP10b
at a common site less than 1200 nt from the potential translational
initiation sites so that the predicted 20-amino acid difference between
the translation products of P-CIP10a and P-CIP10b would be detectable
(Fig. 2B). Each P-CIP10 cDNA yielded a protein of the
size predicted if translation were initiated at the Met in each unique
5
-region (Fig. 2C); for both P-CIP10a and P-CIP10b, the
next Met is more than 80 amino acid residues downstream. The P-CIP10a
transcription/translation reaction proceeded much more efficiently than
the P-CIP10b reaction, and we used the P-CIP10a cDNA for all
further studies.
P-CIP10a encodes a protein of 1899 amino acids with a calculated molecular mass of 217 kDa and pI of 5.67 (Fig. 2D). P-CIP10 is largely hydrophilic, with the characteristics of a cytosolic protein. The NH2 terminus of P-CIP10 lacks a hydrophobic signal sequence, and no hydrophobic stretches typical of transmembrane domains are present. By homology search and computer-based structural analysis, P-CIP10 can be divided into five regions: a short NH2-terminal region, a region of spectrin-like repeats, a DH domain, a PH domain, and the COOH-terminal region (Fig. 2E).
The NH2-terminal 150 amino acids of P-CIP10 are homologous
to Trio, a new member of the Dbl family of proteins identified by
virtue of its interaction with the cytosolic domain of the leukocyte
common antigen-related (LAR) transmembrane protein-tyrosine phosphatase
(33). The next 1000 amino acid residues of P-CIP10 are most homologous
to Trio (33), spectrin, and fodrin (34, 35). Spectrin and fodrin are
cytoskeletal proteins involved in the maintenance of plasma membrane
structure by cross-linking to actin and to various integral and
membrane-associated proteins (36, 37). Secondary structure predictions
indicate that this region of P-CIP10 is almost entirely -helical and
that the NH2-terminal part of P-CIP10 can be arranged into
nine spectrin-like repeats 103-138 amino acids in length (Figs.
2E and 3A).
Separated from the spectrin-like repeats by 50 amino acids is a 200-amino acid region (aa 1258-1457) with significant homology to the DH domain defined by Dbl, Dbs, and Ost (24, 38-40) (Fig. 2, D and E, and Fig. 3B). DH domains were identified first in a family of oncogenic proteins and subsequently shown to catalyze the exchange of bound GDP for bound GTP on specific members of the Rho subfamily of small GTP binding proteins (24). The DH domain with the highest homology to the DH domain of P-CIP10 is the first DH domain of human Trio (90% identity) (Fig. 3B). The DH domains of Dbl, Ost, and Dbs share 42-46% identity with that of P-CIP10. Additional proteins that share significant homology with the DH domain of P-CIP10 include Tiam, FGD1, and yeast SCD1 (Fig. 3B) (41-43).
The region of P-CIP10 immediately following the DH domain (Fig. 2, D and E; aa 1458-1555) constitutes a PH domain. PH domains are poorly conserved in sequence and are defined by their common three-dimensional structural motifs (44, 45). The PH domain of P-CIP10 has greatest similarity to the PH domain of Trio, followed by the PH domains of Dbl, Dbs, and Ost (Fig. 3B). Although not essential for in vitro GEF activity, the PH domain is generally essential to cellular function (46). PH domains are thought to aid in protein localization by protein-protein or protein-lipid interaction (44, 45, 47).
Except at its extreme COOH terminus, P-CIP10 exhibits homology to Trio. The COOH-terminal third of the 2861-amino acid Trio protein contains a second DH/PH domain, immunoglobulin-like repeats, and a putative serine/threonine protein kinase domain and these last two domains bear no homology to P-CIP10. Since the human ESTs identified as homologues of P-CIP10 (Fig. 2D) exhibit a greater degree of identity to rat P-CIP10 than does human Trio, we conclude that Trio and P-CIP10 are encoded by separate genes. The sequence homology exhibited by P-CIP10 and Trio suggests that these two proteins define a subfamily of the Dbl proteins.
Expression of P-CIP10 in AtT-20 CellsTo study the properties of P-CIP10 and its effects on cellular function, P-CIP10a and Myc.P-CIP10 cDNAs (Fig. 2A) were used to doubly transfect AtT-20 cells stably expressing PAM-1 (AtT-20/PAM-1 cells). AtT-20 corticotrope tumor cells do not express P-CIP10 at a level that allows detection by Northern blot analysis of total RNA (data not shown). Although the transfected P-CIP10 mRNA could easily be visualized by analyzing 10 µg of total RNA, the P-CIP10 protein proved difficult to detect. At least two clonal cell lines expressing P-CIP10 and two expressing Myc.P-CIP10 were selected based on Northern blot analysis and one of each was studied in detail.
Direct visualization of P-CIP10 proved impossible with available
antisera, so we concentrated the Myc.P-CIP10 protein by
immunoprecipitation. Extracts of AtT-20/PAM-1 cells expressing
Myc.P-CIP10 were adsorbed to Myc monoclonal antibody immobilized on
Protein G beads. Bound proteins were subjected to Western blot analysis
and visualized with a rabbit polyclonal antiserum to P-CIP10 (aa
447-1138) (17) (Fig. 4A). A cross-reactive
protein of 210 kDa was detected in cells expressing Myc.P-CIP10 but not
in AtT-20/PAM-1 cells.
Expression of P-CIP10 in AtT-20 cells could also be demonstrated by metabolic labeling and immunoprecipitation. AtT-20/PAM-1 cells expressing P-CIP10, Myc.P-CIP10, or only PAM-1 were incubated in medium containing [35S]Met/Cys for 15 min, extracted, and subjected to immunoprecipitation using antibody to recombinant GST.P-CIP10 (aa 447-1138) (Fig. 4B). A radiolabeled protein of 210 kDa was detected in both P-CIP10 lines and accounted for 0.03 ± 0.01% of the total protein synthesized during a 30-min pulse. The fact that P-CIP10 was more easily identified using metabolic labeling methods suggested that the protein might have a short half-life. Using a pulse/chase paradigm, both P-CIP10a and Myc.P-CIP10 were found to turn over quickly (Fig. 4C). A semilogarithmic plot of the densitized band intensities yielded a half-life estimate of 60 min for both proteins (Fig. 4D).
To localize P-CIP10, AtT-20/PAM-1 cells expressing Myc.P-CIP10 were biosynthetically labeled for 30 min and subjected to differential centrifugation (Fig. 4E). PAM-1 is recovered in fractions enriched in endoplasmic reticulum (P1 and P2) as well as in fractions enriched in TGN (P2 and P3) and secretory granules (P3). Each particulate fraction as well as the cytosolic fraction contained Myc.P-CIP10. The association of P-CIP10 with particulate fractions, many of which contain intact PAM-1 (23), suggests that P-CIP10 may function by interacting with membranous organelles.
Expression of P-CIP10 Changes AtT-20 Cell MorphologySince
many members of the Dbl family of proteins interact with members of the
Rho family of GTPases and affect cytoskeletal organization and cell
shape (48, 49), we examined the morphology of our stably transfected
AtT-20 cells (Fig. 5). Many cells expressing P-CIP10
were larger than wild type AtT-20 or AtT-20/PAM-1 cells. In addition,
cells expressing P-CIP10 often had very long processes, some of which
were branched. Photomicrographs of randomly selected fields of each
cell type were analyzed; the number of giant cells, the percentage of
cells having neuritic processes, and the lengths and shapes of
processes were quantified (Fig. 5E). Approximately 15% of
the total population of P-CIP10-expressing cells had greatly enlarged
cell bodies, 5-6 times more than for nontransfected as well as for the
PAM-1-expressing cells. Twice as many of the P-CIP10-expressing cells
had processes, although the average number of processes per cell for
cells with processes was unaltered. The percentage of branched
processes and the percentage of processes longer than 100 µm were
dramatically increased for the cells expressing P-CIP10.
Expression of P-CIP10 Alters the Metabolism of PAM-1
In AtT-20 cells, the 120-kDa PAM-1 protein is cleaved by neuroendocrine-specific endoproteases after it exits the TGN (50). Cleavage yields soluble 45-kDa PHM and a 70-kDa membrane protein containing the PAL, transmembrane, and COOH-terminal cytosolic domains. Active, 45-kDa PHM is stored in large dense core vesicles from which its secretion can be regulated by secretagogues. Membrane PAM proteins that are localized to the cell surface can be cleaved at a site near the transmembrane domain, leading to the accumulation of bifunctional 105-kDa PAM in the medium (8).
To determine whether expression of P-CIP10 changed the metabolism of
PAM-1, we performed pulse/chase metabolic labeling experiments on
AtT-20/PAM-1 cells and AtT-20/PAM-1 cells expressing P-CIP10a or
Myc.P-CIP10. Quadruplicate wells of each cell type were incubated in
medium containing [35S]Met/Cys and either harvested
immediately (P; pulse) or incubated in the presence of
unlabeled methionine/cysteine for 1, 2, or 4 h (C;
chase) (Fig. 6). Full-length PAM-1 (120 kDa) disappeared more rapidly from cells expressing P-CIP10 than from PAM-1 cells (Fig.
6A). Consistent with this, production of 45-kDa PHM occurred at a slightly earlier time in cells expressing P-CIP10 (Fig.
6A; 45 kDa). The total amount of PAM protein recovered after
4 h of chase was less in AtT-20/PAM-1 cells expressing P-CIP10.
More bifunctional 105-kDa PAM accumulated in the medium of cells
expressing PAM-1 than in the medium of cells expressing PAM-1 and
P-CIP10 (Fig. 6B; 105 kDa).
P-CIP10 Interacts with Rac1 GTPase
The presence of a DH
domain followed by a PH domain in P-CIP10 strongly suggested that it
would interact with members of the Rho subfamily of small GTP-binding
proteins (48, 49). The GST fusion proteins of Rac1, RhoA, and Cdc42
were depleted of nucleotide, bound to glutathione-Sepharose, and
incubated with extracts of AtT-20 cells expressing PAM-1 or
PAM-1/Myc.P-CIP10. The bound proteins were analyzed by Western blot or
by immunoprecipitation. Extracts of AtT-20 PAM-1/P-CIP10 cells
contained a 210-kDa protein that bound to Rac1 but not to RhoA or Cdc42
(Fig. 7). Binding occurred in the absence of bound
nucleotide and was not observed when extracts of control AtT-20 PAM-1
cells were used. We conclude that P-CIP10 is capable of interacting
specifically with Rac1.
Since our previous mutagenesis studies identified the CD of membrane PAM as essential in establishing its steady state localization in neuroendocrine cells, we screened a rat hippocampal cDNA library for PAM CD interactors using the yeast two-hybrid system (17). We previously identified partial cDNAs encoding a putative protein serine/threonine kinase (P-CIP2) and a protein with spectrin-like repeats (P-CIP10). Elucidation of the complete structure of P-CIP10 revealed the presence of elements common to signal transduction pathways, and expression of P-CIP10 in AtT-20 cells altered PAM processing and cell morphology. The itinerary taken by membrane PAM proteins in AtT-20 cells is complex, and it is anticipated that additional proteins capable of interacting with the CD of PAM will be identified.
The expression of P-CIP10 is highest in specific areas of rat brain.
PAM is expressed in the same areas, but PAM is also expressed at high
levels in many tissues lacking P-CIP10. For example, atrium, anterior,
and neurointermediate pituitary express little P-CIP10 but high levels
of PAM. P-CIP10 may fulfill a function unique to its sites of
expression, or homologous protein(s) may be involved in the routing of
PAM in tissues lacking P-CIP10. The existence of P-CIP10 transcripts
with different 5-ends and different sizes suggests that alternate
splicing generates diverse forms of P-CIP10. Routing signals in the CD
of PAM are recognized in both neuroendocrine and nonneuroendocrine
cells (7, 8), and proteins homologous to P-CIP10, but expressed more
widely, may be involved in these interactions.
The co-expression of P-CIP10 and PAM-1 in AtT-20 cells increased the rate and extent of disappearance of newly synthesized PAM-1. After traversing the Golgi stacks, newly synthesized membrane PAM exits the TGN and enters immature secretory granules (50). Although some membrane PAM remains in mature secretory granules, most of the membrane PAM leaves the immature granules, perhaps via constitutive-like vesicles. Membrane PAM that reaches the cell surface is efficiently internalized, with mutagenesis studies suggesting that access to lysosomes is affected by phosphorylation (23). The phenotype observed suggests that P-CIP10 affects routing of PAM in the TGN/immature secretory granule region of the cell. P-CIP10 protein, which turns over with a half-life of less than 1 h, was recovered in particulate fractions and in cytosol, and factors affecting its interaction with particulate fractions remain to be identified.
The structure of P-CIP10, a member of the growing Dbl family of proteins, provides a great deal of insight into its possible functions (24, 51). The first members of this family, Dbl, Dbs, and Ost, were identified by their oncogenic activity. Family members share DH and PH homology domains, and most have been shown to serve as GDP/GTP exchange factors for specific members of the Rho family of small GTP-binding proteins. Oncogenic forms of Dbl family members often lack NH2-terminal regulatory domains, and the effect of expressing P-CIP10 lacking this region remains to be investigated. Trio, which is broadly expressed, is most closely related to P-CIP10 (33). Trio was identified by virtue of the interaction of its COOH-terminal domain with the cytosolic domain of the LAR receptor type protein-tyrosine phosphatase (33); characterized forms of P-CIP10 lack a homologous region. Phogrin, a homologue of the LAR protein-tyrosine phosphatase, is localized to the regulated secretory pathway of neuroendocrine cells (52), and several other receptor type protein-tyrosine phosphatases are neuroendocrine-specific, raising the possibility that they interact with P-CIP10-related molecules (53-55).
The NH2-terminal half of P-CIP10 contains 9 units
homologous to the 5-nm-long structural units that make up spectrin
(68). Spectrins provide structural support to the plasma membrane and form an extensive cytoskeletal meshwork by binding to specific soluble
and integral membrane proteins (36, 37, 56). The spectrin-based
membrane skeleton restricts the mobility of membrane proteins. An
isoform of -spectrin associated with Golgi membranes in Madin-Darby
canine kidney cells may play a role in sorting proteins into the
vesicular transport pathway (57). The fact that P-CIP10 expression
facilitates cleavage of newly synthesized PAM by enzymes located in
immature secretory granules identifies the TGN/immature secretory
granule as a subcellular site at which PAM/P-CIP10 interactions
occur.
The DH/PH domain of P-CIP10 is most homologous to the first DH/PH domain of human Trio (33). Consistent with this homology, P-CIP10 binds to Rac1 but not to RhoA or Cdc42. Many members of the Dbl family, through their DH/PH domain, act as GDP/GTP exchange factors for members of the Rho family of small GTP binding proteins and play important roles in regulating cytoskeletal organization (49, 58). The ability of P-CIP10 to act as a GDP/GTP exchange factor remains to be tested. The PH domain of known Dbl family members is generally not essential for in vitro GDP/GTP exchange factor activity, but it is essential for cellular function (46, 59). PH domains and the structurally related phosphotyrosine binding domains can bind specific lipids, phosphopeptides, or proteins, targeting the neighboring DH domain to the proper subcellular location (45, 46, 60). The COOH-terminal region of P-CIP10 retains some homology to the corresponding region of Trio but shows no homology to other proteins in the data base.
Expression of P-CIP10 in AtT-20 cells was associated with the presence of more giant cells, more cells with neuritic processes, and more long processes that branched. Although the underlying mechanisms are not clear, the fact that P-CIP10 interacts with Rac1 is consistent with the occurrence of cytoskeletal effects. In Drosophila, Rac1 affects axonal but not dendritic outgrowth (61), and expression of constitutively active Rac1 in the Purkinje cells of transgenic mice alters both axonal and dendritic morphology (62). In fibroblasts, Rac1 regulates formation of lamellipodia and membrane ruffling (49). Rac1 has been shown to interact with a variety of protein and lipid kinases, thus affecting actin cytoskeletal organization, transcriptional activation, cell proliferation, and secretion (49, 58, 63, 64). Studies using perforated cells demonstrated an inhibitory role for Rac and Rho in transferrin receptor-mediated endocytosis through coated pits (65), and kinectin, a vesicle membrane anchoring protein for kinesin binds activated Rac (66). Overexpression of Tiam1 or activated Rac1 in T-lymphoma cells induced invasiveness (67).
Perhaps most intriguing about the identification of a Dbl family member as a PAM CD interactor is the possibility that we have identified a signal transduction pathway linking the lumen of the secretory pathway to the cytosol. P-CIP10, a putative GDP/GTP exchange factor, interacts with integral membrane PAM, whose pH and conformation-sensitive functional domains (27) reside in the lumen of the regulated secretory pathway, and with Rac1, a small GTP-binding protein that is a component of several distinct cytosolic signal transduction pathways. These interactions suggest a relationship between large dense core vesicle biogenesis and the signal transduction machinery. Identification of additional large dense core vesicle and cytosolic interactors with P-CIP10 and studies on the effects of P-CIP10 on signal transduction pathways regulated by Rho-like GTPases should provide a mechanistic understanding of the biogenesis and routing of large dense core vesicles. We propose giving P-CIP10 the name kalirin to signify its ability to interact with multiple proteins (Kali, Hindu goddess with many hands) and its spectrin-like domains.
We thank Dr. Richard Cerione and Dr. Shubha Bagrodia (Cornell University) for providing the GST-Rac1, RhoA, and Cdc42 expression vectors and Dr. Henry Keutmann (Massachusetts General Hospital) for synthesizing the P-CIP10 peptide. We thank Marie Bell for helping with all aspects of this work and Kate Deanehan for help developing the monoclonal antibody.