(Received for publication, October 26, 1994; and in revised form, December 2, 1994)
From the
SH2 and SH3 domains have been characterized as functional domains that mediate protein-protein interactions in signal transduction. Recently, the cDNA sequence of a novel Src- and Fyn-binding protein called AFAP-110, for Actin-Filament Associated Protein-110 kDa, was reported. This protein was distinctive in that it is both an SH2 and SH3 binding partner for the non-receptor tyrosine kinases Src and Fyn. Here, we report the characterization of an alternatively processed form of AFAP-110 that encodes an additional 258 base pair (bp) of open reading frame. Transient expression of this full-length clone reveals a molecular mass of 120 kDa. Western blot analysis indicate that a larger 120-kDa variant of AFAP-110 can be detected in brain and is not detectable in any other tissues examined. Northern blot analysis indicate that the novel 258-bp insert can be detected in brain RNA but not chick embryo fibroblast RNA. We propose the name AFAP-120, for Actin Filament-Associated Protein-120 kDa. Expression of the 258-bp novel insert (NINS) as a glutathione S-transferase-encoded fusion protein permits adsorption of a 67-kDa protein from tissue lysates. Deletion analysis of the NINS indicates that the interaction with p67 can be attributed to a proline-rich motif that resembles an SH3-binding motif. We hypothesize that AFAP-120 facilitates interactions in brain between SH2/SH3 signaling proteins and actin filaments and that a proline-rich motif in the NINS may exist to facilitate additional interactions between cellular proteins in brain and actin filaments.
SH2 and SH3 domains are recognized as conserved functional
domains found in a variety of proteins known to play an important role
in signal transduction, including the src family of
nonreceptor tyrosine kinases, phospholipase C-, and
phosphatidylinositol 3-kinase(1) . The SH2 domain mediates
protein-protein interactions by recognizing and binding to peptide
motifs that contain a phosphotyrosine residue(1) . The function
of the SH3 domain is less clear(2) . This domain, unlike the
SH2 domain, is also found in proteins known to interact with the
cytoskeleton and cell membrane(3, 4) . Hence, the SH3
domain has been hypothesized to mediate protein-protein interactions
with these cellular structures(1) . Recent studies indicate
that the SH3 domain may also serve to link proteins to ras signaling
pathways(5, 6, 7, 8) . Evidence for
this association was originally pointed out in the cloning of the abl SH3-binding protein, 3BP-1(9). This polypeptide contains
sequences distinct from its SH3-binding motif which are homologous to
the family of GTPase activating proteins that act upon the rac/rho family of small G-proteins. Additionally, recent evidence
indicates that rac and rho control membrane ruffling
and stress fiber organization, respectively(10, 11) .
Thus, these data indicate a link between SH3-containing proteins, ras signaling pathways, and cytoskeletal associations.
Recently, we described the predicted amino acid sequence of a novel
Src-binding protein, called AFAP-110(12) . This novel
cytoskeletal-associated protein was distinctive in that it could be
independently adsorbed with bacterial encoded fusion proteins
expressing either the SH2 or SH3 domains of pp60 or pp59
(12) . Furthermore, AFAP-110
encodes predicted SH2- and SH3-binding motifs(12) . These data
indicate that AFAP-110 is both an SH2- and SH3-binding partner for Src
and that it is capable of forming a stable interaction with more than
one member of the Src family of nonreceptor tyrosine kinases. It was
hypothesized that one possible function of AFAP-110 was to facilitate
interactions between SH2/SH3-containing proteins and the
cytoskeleton(12) . These stable complexes may be significant as
the binding of AFAP-110 to activated forms of Src correlate strongly
with transformation(13, 14) . Activated variants of
Src that contain deletions in the SH2 or SH3 domains are both defective
for transformation and unable to form a stable complex with
AFAP-110(13, 14) . These data indicate that one
criterion for transformation would require intact SH2 and SH3 domains
to facilitate interactions with SH2 and SH3 cellular binding partners.
Because AFAP-110 is associated with actin
structures(12, 13, 15) , and the concomitant
disruption of actin cables is a hallmark of transformation by
Src(16, 17) , then AFAP-110 could play an important
role in modulating the effects of Src on the cytoskeleton. In addition,
AFAP-110 may represent an important component in the pathway of signal
transduction through c-Src and c-Fyn.
In this report, we describe
the isolation and characterization of a variant form of the Src
SH2/SH3-binding protein AFAP-110. This larger variant, called AFAP-120,
has a molecular mass of 120 kDa and encodes a novel domain containing
86 amino acids placed near the carboxyl terminus which do not disrupt
the downstream reading frame homologous to AFAP-110. Northern and
Western blot analysis demonstrate that this larger variant can be
detected in brain. AFAP-120 retains predicted SH2- and SH3-binding
motifs and can be efficiently adsorbed by GST-SH3 created
from c-Src. The novel 86-amino-acid insert encodes a unique sequence
that contain a proline-rich region resembling an SH3-binding motif.
Expression and immobilization of the NINS as a GST-encoded fusion
protein (GST-NINS) reveals that a 67-kDa protein from brain lysates
will associate with GST-NINS, but not to GST alone. This association
requires the integrity of a proline-rich motif in the NINS. We
hypothesize that AFAP-120 may be an important binding partner for c-Src
in brain and that the NINS may facilitate additional interactions
between cellular proteins and actin filaments.
The pCMV-1 vector was obtained from the laboratory of Dr. E. H. Humphries and was used to express cloned proteins in COS-1 cells via the CMV promoter(25) . The cDNAs encoding AFAP-110 and the FLC2.1 were excised from pBluescript ks+ with HincII and SmaI digestion and subcloned into pCMV-1 via the unique SmaI restriction site in the multiple cloning site.
The novel 258-bp insert is
surrounded by sequence that define Sau3AI sites. This Sau3AI fragment encodes Ser to Ser
and does not include the three carboxyl-terminal amino acids of
the NINS that predict Val
, Ser
, or
Gln
. This sequence was subcloned into the novel BamHI site in pGEX-2T and transfected into DH5-
bacteria.
This GST-encoded fusion protein (GST-NINS for NINS) was
expressed as described previously (12) . The preparation of
bacterial lysates containing these fusion proteins, and immobilization
on glutathione-Sepharose beads, were done according to the method of
Smith and Johnson(29) . Immobilized GST fusion proteins were
incubated with 500 µg of chick embryo day 16 brain tissue lysate
for 1.5 h at 4 °C. The bound proteins were washed two times with
RIPA and two times with Tris-buffered saline, pH 7.5. Adsorbed brain
tissue proteins were removed by incubating the beads in boiling 2
Laemmli sample buffer for 2 min, centrifuging, and resolving
the supernatant by 8% SDS-PAGE. The resolved brain lysate proteins were
analyzed by ammoniacal silver staining according to the method of
Harlow and Lane(32) .
Figure 1:
Analysis of clone p2.1 and construction
of a full-length version of clone p2.1. A, clone p2.1 was
sequenced on both strands. The cDNA sequence of the NINS was isolated
by Sau3AI digestion and used to isolate two additional clones,
p8.1 and p6.1, which were sequenced completely on one strand. Clones
p8.1 and p6.1 encode the NINS, as well as sequence identical to
AFAP-110 and clone p2.1. A full-length version of clone p2.1 (FLC2.1)
was created by subcloning the BglII to BglII fragment
of clone p2.1, which encodes the novel 258-bp insert, into the cDNA
encoding AFAP-110. This BglII to BglII fragment
encodes Leu to Asp
, predicted for clone
p2.1. B, the predicted amino acid sequence of AFAP-120 from bp
1453 (common to AFAP-110 and AFAP-120) to bp 1902 (bp 1644 for
AFAP-110). The cDNA sequence of the novel 258-bp insert is underlined, along with the predicted amino acid sequence.
Sequence information that is not underlined represents sequence common
to both AFAP-110 and clone p2.1. A proline-rich region is highlighted in the box defining Pro
to
Pro
.
The 258-bp insert encodes 86 amino
acids and retains the integrity of the serine residue encoded at amino
acid position 510 (Fig. 1B). In addition, the 258-bp
insert does not disrupt the downstream reading frame predicted from the
AFAP-110 encoded phenylalanine 511 (Fig. 1B).
Interestingly, a second variant cDNA has been isolated that diverges
from AFAP-110 cDNA sequence at exactly the same bp (bp 1549); however,
this variant encodes 19 novel amino acids after serine 510, followed by
an opal stop codon and 3`-noncoding sequence. ()Because two
different variants of AFAP-110 have been isolated that diverge at
exactly the same site, we speculate that AFAP-120 may have arisen by
alternative splicing. The predicted amino acid sequence of the 258-bp
insert revealed a novel sequence that was not homologous with any
proteins in the GenBank data base. The 86-amino-acid sequence contains
a disproportionate number of serine residues (15 serine residues, or
17% of the insert) and a short proline-rich motif (boxed). To
characterize the NINS, a full-length version of this variant was
created by substituting the BglII to BglII fragment
of p2.1 (bp 1162-2056) into the same BglII to BglII restriction site of full-length AFAP-110 (Fig. 1A). This clone is referred to as full-length
clone p2.1 (FLC2.1) and encodes 723 amino acids with a predicted
molecular mass of 81,458 Da.
Figure 2: Detection of a 120 kDa form of AFAP-110 in chick brain. Twenty-five µg of tissue lysates were diluted with RIPA to 1 µg/µl and resolved by 8% SDS-PAGE. The cellular proteins were transferred to nitrocellulose by Western transfer and probed for the presence of AFAP-110 using mAb 4C3. Tissues include CE cells (lane 1), brain (lane 2), eye (lane 3), heart (lane 4), lung (lane 5), muscle (lane 6), kidney (lane 7), liver (lane 8), intestine (lane 9), bladder (lane 10), spleen (lane 11). The data are representative of three independent experiments.
Figure 3: Expression of FLC2.1 and AFAP-110 in COS-1 cells. Twenty-five µg of day 16 chick embryo brain lysate (lane 1), CE lysate (lane 2) and COS-1/pFLC2.1 (lane 3), COS-1/pAFAP-110 (lane 4), or COS-1/pCMV-1 (lane 5) transfected COS-1 cell lysates were resolved by 8% SDS-PAGE, Western transferred, and probed for the presence of AFAP-110 or AFAP-120 with mAb 4C3. Reactive proteins were quantitated with sheep anti-mouse antibodies conjugated to horseradish peroxidase and detected by chemiluminescence. Lanes 1 and 2 represent a 2-min exposure, while lanes 3-5 represent a 10-s exposure. The data are representative of three independent experiments.
Interestingly, an immunoreactive polypeptide of 92 kDa is detected in pCMV-FLC2.1-transfected COS-1 cells and an 82-kDa immunoreactive polypeptide is detected in pCMV-AFAP-110-transfected COS-1 cells, (Fig. 3, lanes 3 and 4, respectively). It is likely that these lower molecular weight forms are the products of proteolytic degradation. Similarly sized polypeptides are detected in cell and tissue lysates (see Fig. 2) and have been described earlier and postulated to be proteolytic degradation products(13) .
Figure 4: Northern blot analysis. Twenty µg of total RNA was isolated from chick brain and CE, resolved on a 1.5% agarose gel, transferred to Immobilon, and probed with probe A for the presence of sequence common to AFAP-110 and AFAP-120 (lanes 1 and 2) or with probe B to detect the novel 258-bp insert (lanes 3 and 4).
Interestingly, it was noted that a
proline-rich region which resembles an SH3-binding motif is encoded in
the NINS between amino acids 541 and 551(23) . This
proline-rich motif exists in a region of the NINS that has a high
hydrophilicity prediction. It has already been established
that AFAP-110 has two potential SH3-binding motifs in the amino
terminus and that AFAP-110 is an SH3-binding partner for Src and
Fyn(12) . Because it has been hypothesized that the function of
AFAP-110 is to facilitate interactions between actin filaments and SH2-
and/or SH3-signaling proteins, it was predicted that the NINS might
facilitate interactions with additional cellular proteins. To test this
hypothesis, the NINS was subcloned into pGEX-2T by isolating a 247-bp
fragment that defines the 5` and 3` borders of the insert and
subcloning it into the novel BamH I site of pGEX-2T. GST-NINS
and GST were used to adsorb potential binding proteins from day 16
chick embryo brain lysates, and the molecular mass of these proteins
evaluated by silver stain. To evaluate specificity in binding, three
deletions were constructed in NINS (Fig. 5A). A StuI-SmaI deletion eliminates amino acids
536-593 predicted within the NINS. This binding protein is
referred to as GST-NINS
and encodes the first 25
amino acids of the NINS (Ser
to Leu
). In
addition, the sequences encoding Leu
to
Ser
, or Asp
to Ser
, were
subcloned into pGEX-2T and pGEX-1, respectively. These GST fusion
proteins, GST-NINS
and
GST-NINS
, encode the carboxyl-terminal 55 or 41
amino acids of the NINS, respectively. These data reveal that a 67-kDa
protein is adsorbed by GST-NINS (Fig. 5B, lane
1) and GST-NINS
(Fig. 5B, lane 4), but not with GST or GST-NINS
(Fig. 5B, lanes 2 and 5,
respectively). These data indicate that the amino acid sequence between
Tyr
and Asp
, which encodes the proline-rich
motif, is critical for binding p67. There appears to be a small amount
of a 67-kDa protein absorbed by GST-NINS
(Fig. 5B, lane 4), indicating that
sequence interactions defined between Ser
and Leu
may also contribute to binding.
Figure 5:
Description of a potential cellular
binding partner for the NINS. A, GST-NINS encodes Ser to Ser
. Three deletion variants of GST-NINS were
constructed. GST-NINS
encodes the first 25 amino
acids of the NINS (Ser
to Gly
).
GST-NINS
encodes Leu
to
Ser
. GST-NINS
encodes Asp
to Ser
. B, 250 µg of chick brain
lysate (from a day 9 embryo) were preincubated with GST immobilized on
Sepharose beads, precleared, and then incubated with GST-NINS (lane
1), GST (lane 2), GST-NINS
(lane
3) GST-NINS
(lane 4), or
GST-NINS
(lane 5). Adsorbed brain lysate
proteins were eluted with boiling Laemmli sample buffer, resolved by 8%
SDS-PAGE, and analyzed by silver stain. The abundant protein bands
between 26 and 36 kDa represent the GST-encoded fusion proteins. These
data are representative of three independent
experiments.
GST-NINS and
GST-NINS were used to probe other avian tissue
lysates to determine whether there might be specificity in the
interaction between p67 and the NINS.
The data indicate
that an equivalent amount of p67 can be adsorbed from brain, lung,
muscle, liver, and kidney. Theses data support a hypothesis that p67
may not be a specific binding partner for the NINS. However, the data
would indicate that the proline-rich region contained within the NINS
may be responsible for facilitating protein-protein interactions in the
brain. It is likely that p67 contains a structure that is favorable for
interacting with the NINS proline-rich motif. Taken together, these
data indicate that the function of the NINS might be to facilitate
protein-protein interactions in brain via an internal proline-rich
motif.
Ren et al.(23) predicted that a consensus SH3-binding motif would be approximately 10 amino acids long and contain proline residues at peptide positions 2, 7, and 10 that would be critical for binding. In addition, a proline residue at position 9 is frequently identified as a component of an SH3-binding motif, and an alanine residue at position 1 appears to be important for binding. Lastly, a hydrophobic amino acid at peptide position 8 may contribute to stability in SH3 binding. The NINS contains a region of 11 amino acids that are almost identical to the consensus SH3-binding motif predicted by Ren et al.(23) . This sequence contains proline residues at peptide positions -1, 2, 9, and 10, as well as a hydrophobic tyrosine residue at position 8 and serine at position 1 (a conserved change). However, this stretch of amino acids contains an arginine residue rather than a proline at peptide position 7. A proline residue at peptide position 7 was identified as being very important for stable SH3 binding(23) . This variation could be required for some specific interaction with another cellular protein. Because the proline-rich region in the NINS resembles an SH3-binding motif, it is possible this structure could facilitate additional protein-protein interactions. It is also possible that the function of the NINS is not related to facilitating protein-protein binding. However, given that the hypothesized function of AFAP-110 is as an adaptor molecule that facilitates interactions between SH2- and/or SH3-containing proteins with the cytoskeleton(12) , it would not be unrealilistic to hypothesize that the NINS may contribute a similar function to AFAP-120, thus enhancing its diverse interactions between cellular proteins and the cytoskeleton.
The function of c-Src has not been discerned; however, it is likely that this proto-oncogene has a specialized function within the brain. This hypothesis is based on several observations which indicate that (a) the c-Src tyrosine kinase is activated in brain(34, 35, 36, 37, 43) ; (b) the c-Src proto-oncogene product is developmentally regulated in neurons, being expressed initially at the onset of neuronal differentiation and then maintained at high levels in fully differentiated neurons in the adult central nervous system(21, 22, 37, 38, 39, 40, 41, 42) ; (c) this proto-oncogene product is alternatively spliced only in neurons(20, 44) ; and (d) upon activation, is capable of inducing neuronal differentiation in cultured neurons, indicating a role in the signal transduction processes that modulate neuronal differentiation(19) . Thus, signaling by c-Src in the brain may be specific and significant.
SH2/SH3 interactions between
AFAP-110 or AFAP-120 and c-Src may represent an important link in
signal transduction by Src. It is possible that pp60 may have a unique function in affecting the integrity of the
cytoskeleton(15, 16, 17, 45, 46, 47, 48) .
Localization studies in neurons indicate that pp60
is
enriched in growth cones, a structure that is also rich in actin
filaments(49, 50) . Further, pp60
isolated from these growth cones have a high tyrosine-specific
kinase activity(49) . Thus, pp60
may have a
specialized function in growth cone extension, which would not be
inconsistent with previous observations that demonstrate a role for
activated forms of Src in modulating cell-cell contacts or
cell-substratum contacts.
One potential function for AFAP-120 would
be to facilitate interactions between SH2/SH3-containing proteins and
the cytoskeleton. AFAP-120 does retain an association with actin
structures, as detected by immunofluorescence with Ab F1 in COS-1 cells
expressing FLC2.1. The function of AFAP-120 in signal
transduction is not clear; however, given that nonreceptor tyrosine
kinases like pp60
and pp59
are
expressed at relatively high levels in brain(18) , it would
appear likely that AFAP-120 would be positioned to interact with, and
possibly mediate a signal transduction event through, these tyrosine
kinases. Lastly, the NINS may play a unique role in facilitating a
similar interaction with other cellular proteins. These stable
complexes may play a functionally important role in modulating the
integrity of actin filaments in response to specific cell signaling
events in the brain.
This work is dedicated to the late Dr. Eric H. Humphries, whose input and critiques were instrumental toward the development of this project.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L20302[GenBank].