ASAP1 was purified and cloned on the basis of being an Arf GAP that was PIP2
dependent and, therefore, a potential link between the phosphoinositide signalling pathway and membrane traffic. ASAP1 was independently identified by screening for proteins that interact with another signalling molecule, the cytoplasmic tryosine kinase Src that is involved in the regulation of cell growth and cytoskeletal organization. ASAP1 contains a GCS1-type zinc finger, the only region of homology with ArfGAP1. In addition, ASAP1 contains a type II SH3 binding site that mediates interaction with Src and a number of other conserved domains that frequently occur in components of established signal transduction pathways. These include a PH domain, an SH3 domain, ANK repeats, and several proline-rich SH3-binding motifs (9
). Based on our data, it is possible that ASAP1 activity or localization is regulated by Src. Furthermore, through interaction with Src as well as phosphoinositides, ASAP1 could coordinate membrane traffic with other cellular responses mediated by these signalling molecules.
Many studies have demonstrated Src’s involvement in regulating cytoskeletal architecture and cell adhesion. Evidence for a role for Src in regulating membrane traffic is accumulating as well. Src has been localized to several membrane compartments in the secretory and endocytic pathways, including endocytic, exocytic, and synaptic vesicles (5
). The activities of Src and its close relative Fyn are modulated during secretagogue-stimulated exocytosis of chromaffin cells (2
), and tyrosine kinase activity is required for exocytosis (18
). Src stimulates epidermal growth factor receptor internalization (95
). Src associates with or phosphorylates various proteins implicated in vesicle transport, including synapsin I, dynamin, synaptophysin, synaptogyrin, and cellugyrin (5
). Like ASAP1, synapsin I and dynamin bind Src through the Src SH3 domain, but neither protein is phosphorylated on tyrosine (31
). The clathrin adapter protein α-adaptin is also found in Src-dynamin complexes (31
). Synaptophysin, synaptogyrin, and cellugyrin all appear to be Src substrates (5
). Src may also influence membrane traffic by interacting with or activating enzymes involved in phospholipid metabolism such as phosphatidylinositol 3-kinase and phospholipase D (12
). We are currently examining stimuli that activate Src to identify upstream signals that result in ASAP1 phosphorylation.
ASAP1 is broadly expressed and may regulate Arf activity in a variety of cell types. The two cDNA clones isolated, ASAP1a and ASAP1b, are indicative of alternative splicing of the mRNA. The exon deleted in ASAP1b encodes part of the proline-rich Src interaction domain and includes several PXXP motifs that might serve as SH3 binding sites, e.g., 817PTGPPSTLP, 841RPPPPPPGHK (P2), and 856PPSPLPHGPP. However, this exon is not required for binding Src. Both an ASAP1a fragment lacking the 856PPSPLPHGPP sequence and an ASAP1a construct with proline mutations in 841RPPPPPPGHK were able to bind Src family SH3 domains in vitro. Although not important for Src SH3 binding, the ASAP1a-specific exon may have another role.
In addition to interacting with Src in vivo and in vitro, ASAP1 also interacted in vitro with Crk, an adapter protein consisting of one SH2 and two SH3 domains (82
). Mutated or activated forms of Src and Crk cause changes in cytoskeletal architecture that can result in cellular transformation. In addition, Src and Crk associate with some of the same partners, including proteins of cell adhesion signalling pathways such as the focal adhesion proteins p130CAS
and paxillin (reviewed in reference 36
). Since at least two proline-rich sites in ASAP1 contributed to binding Crk and Src SH3 domains, it is possible that both proteins could bind ASAP1 simultaneously or compete for the same sites. The role that Src and Crk interactions with ASAP1 play in ASAP1 localization and activity is currently being investigated.
Arfs are known to function at a number of intracellular sites, including the plasma membrane (25
). Because the first mammalian Arf GAP cloned, ArfGAP1, is confined to the Golgi apparatus (4
), the regulation of Arfs at other sites would require unique GAPs. Consistent with this, several distinct Arf GAP activities have been purified (19
). Of these, ASAP1 is the second mammalian GAP to be cloned and shown to be a gene product distinct from ArfGAP1. ArfGAP1 and ASAP1 are divergent proteins with homology limited to 38% identity over the 86 amino acids that include the Arf GAP domain–GCS-like zinc finger domain (Fig. B). Despite these structural differences, the proteins have similar Arf specificities (77
); therefore, rather than being GAPs for different Arfs, the structural divergence likely provides differential localization and regulation. The different lipid requirements of the native proteins, as previously reported (77
), are consistent with independent regulation, and the immunofluorescence reported here supports the idea of differential localization. In contrast to ArfGAP1, ASAP1 did not colocalize with markers of the Golgi apparatus, and overexpression of ASAP1 had no detectable effect on Golgi morphology (Fig. ). Instead, ASAP1 was found mostly in the cytosol, with a smaller population at the plasma membrane (Fig. ). This localization pattern is consistent with ASAP1 being both a target for Src and regulated by phosphoinositides. ASAP1 could also be an effector for Arf. Because ASAP1 must bind to Arf-GTP, it could transmit a signal from Arf-GTP.
Based on the specificity of ASAP1 in vitro, ASAP1 is expected to use class I and class II Arfs as substrates. The localizations of Arf1, a class I Arf, and Arf6, a class III Arf, have been determined (13
). Arf1 is considered to be Golgi associated, whereas Arf6 is at the plasma membrane; however, a number of studies support a role outside of the Golgi for class I and class II Arfs, including Arf1. In vitro studies have shown that class I Arfs affect diverse processes including intra-Golgi transport, endoplasmic reticulum-to-Golgi transport, endosome-to-endosome fusion, and synaptic-vesicle maturation (23
). In cell fractionation studies, all of the class I and II Arfs have been found associated with endocytic vesicles (96
). In vivo, class I and class II Arfs have been implicated in a number of specialized endocytic events, including synaptic vesicle maturation (6
). We are now testing the endocytic compartment as a possible target site of ASAP1 action. We are also attempting to identify the Arf family member(s) that is the in vivo substrate for ASAP1, which could be restricted by subcellular localization, specific cofactors, or conditions not reproduced in our in vitro assay.
The PH domain of ASAP1 likely contributes to the phosphoinositide-dependent regulation of Arf. Regulation of a number of proteins by phosphoinositide binding to their PH domains has been demonstrated (53
stimulates the activity of ASAP1 and of a recombinant fragment of ASAP1 containing the PH, zinc finger, and ANK repeat domains (PZA). Our preliminary studies suggest that PIP2
binds to the PZA fragment but not to a protein containing only the zinc finger and ankyrin repeats (45
), and this latter protein has no detectable activity. Therefore, the PH domain of ASAP1 may allow phosphoinositide-dependent activation of the Arf GAP domain. However, PIP2
binding to the substrate, Arf, is also important for the GAP reaction (76
). Thus, phosphoinositide likely binds to both the enzyme (PZA) and the substrate (Arf), similar to the dual role of phosphoinositides in regulating the phosphorylation of protein kinase B (88
In the cell, phosphoinositides have complex effects on Arf. Arf has been found to activate both PIP-kinase (29
) and phospholipase D (10
); therefore, the comodulation of GAP activity by PA and PIP2
could be involved in a system of feedforward and feedback loops that control the time that Arf spends in the GTP state (56
). In addition, phosphoinositides contribute to Arf activation. Three Arf exchange factors, ARNO, GRP-1, and cytohesin, contain PH domains and function at the plasma membrane (14
). ARNO has a PIP2
-dependent exchange activity on myristoylated Arf but is able to act on nonmyristoylated Arf independently of PIP2
, suggesting a role for the PH domain and PIP2
in recruiting the enzyme (ARNO) and substrate (Arf) into the same complex (67
). The PH domains of these molecules actually appear to favor binding to PIP3
). In contrast, PIP3
had no effect on ASAP1 activity in preliminary studies (45
), raising the possibility that regulating PIP2
levels could order the inactivation and activation of Arf. Consistent with this idea, insulin-induced PI 3-kinase stimulation causes a PH domain-dependent translocation of ARNO to the plasma membrane (93
). The PH domain of cytohesin, an exchange factor that binds β2 integrin, is also required for membrane recruitment and is essential for PI 3-kinase activation of β2 integrin adhesion (51
In addition to its conserved domains, ASAP1 has an unusual sequence feature, a series of repeats of E/DLPPKP, many of which are separated by the tripeptide QLG. The sequence is repeated eight times, with an additional five degenerate repeats. A database search did not identify other occurrences of tandem repeats of this sequence. Although the repeat region has high proline content and contains seven PXXP sequences, it did not bind any of 10 different SH3 domains tested (11
). One possible role of this domain is to mediate homodimerization of ASAP1 that we have detected in vitro and in vivo (45
ASAP1 has a number of relatives, the closest being the human KIAA0400 protein. The strong sequence similarity and conservation of domain order between mouse ASAP1 and human KIAA0400 suggest they could be species homologs; however, we have three reasons in particular why we think they are different family members. First, using primers complementary to ASAP1 mouse sequence, partial cDNAs were amplified from human and bovine cDNA library by PCR. The predicted amino acid sequences of the human and bovine clones were 99 and 94% identical to ASAP1, respectively (78
). The same region from KIAA0400 is 67% identical to human ASAP1. Second, KIAA0400 lacks the E/DLPPKP repeat region contained in ASAP1. Third, ASAP1 and KIAA0400 sequences diverge in the proline-rich region except for the class II Src SH3 binding sequence. Thus, ASAP1 and KIAA0400 define a new protein family. Both may bind Src, but they could be differentially localized or regulated by interaction with other SH3-containing proteins. There are three additional proteins in GenBank with 40% or greater overall identity to ASAP1 (Fig. B). These proteins, ASAP1, and KIAA0400 all contain a conserved region consisting of, in order from the amino terminus, consecutive PH, zinc finger, and ANK repeat domains. The ordered PZA region, therefore, defines a PZA superfamily of proteins, of which ASAP1 and KIAA0400 constitute a subgroup. Given the diverse sites of Arf action and the number of Arf family members, ASAP1 and other PZA family members could provide site-specific or Arf-specific regulation of membrane traffic.