Arfaptin 1 forms a complex with ADP-ribosylation factor and inhibits phospholipase D.

ADP-ribosylation factors (ARFs) regulate coatomer assembly on the Golgi as well as recruitment of clathrin adapter proteins and are therefore involved in vesicle budding from the Golgi and vesicular transport. They are also regulators of phospholipase D (PLD) activity. Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion. In the present report, we show that arfaptin 1 interacts with 'high speed' membranes independently of ARF. However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi. Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1. These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro.

Arfaptin 1, an ARF-binding protein, inhibits phospholipase D and endoplasmic reticulum/Golgi protein transport.

Class I ADP-ribosylation factors (ARFs) are essential for coatomer and clathrin coat assembly and vesicular transport in the Golgi apparatus. However, little is known about the in vivo regulation of ARF actions. Recently we cloned arfaptin 1, a 39 kDa protein that binds active, GTPgammaS-liganded ARF and translocates with it to Golgi membranes. Here we show that phorbol ester-stimulated phospholipase D (PLD) activity is inhibited in arfaptin 1-overexpressing NIH 3T3 cells and that arfaptin 1 inhibits ARF activation of Golgi-associated PLD. Since PLD activity is thought to play a role in regulating vesicular transport in the secretory pathway, we determined the rate of glycosylation of vesicular stomatitis virus glycoprotein as a measure of protein transport from the endoplasmic reticulum through the Golgi apparatus. Arfaptin 1 overexpression was found to decrease the rate of this reaction approximately two-fold. These data suggest that arfaptin 1 is a regulator of ARF action in the Golgi apparatus.

Phospholipase D mediates matrix metalloproteinase-9 secretion in phorbol ester-stimulated human fibrosarcoma cells.

Phospholipase D (PLD) has been implicated in vesicle trafficking in the Golgi and hence secretion. In this study, we show that the secretion of matrix metalloproteinase-9 (MMP-9) from HT 1080 human fibrosarcoma cells was stimulated by phorbol 12-myristate 13-acetate in a time- and dose-dependent manner that involved protein kinase C. The phorbol ester also increased PLD activity in the cells. Evidence that PLD was involved in the stimulation of MMP-9 secretion was provided by the observations that the secretion of MMP-9 was stimulated by the introduction of short-chain phosphatidic acid (PA) into the growth medium and that inhibition of PA production by 1-propanol inhibited secretion. Using a short-chain diacylglycerol we excluded the possibility that MMP-9 secretion was induced by diacylglycerol formed from PA by phosphatidic acid phosphatase. Furthermore, propranolol, an inhibitor of this enzyme, had no effect on secretion induced by either phorbol 12-myristate 13-acetate or PA. The data presented here indicate that activation of protein kinase C increases MMP-9 secretion in HT 1080 cells and implicate PLD and PA formation in the effect.

Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.

ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.

Ras GTPase-activating protein-associated p62 is a major v-Src-SH3-binding protein.

Oncogenic transformation by v-Src is accompanied by marked morphological changes and cytoskeletal reorganization. Yet, the cytoskeleton-associated proteins with which v-Src interacts are largely unknown. We have studied the binding of v-Src-SH3 domain to cellular proteins utilizing a blot overlay procedure with a GST-v-Src-SH3 fusion protein as probe. A major 62-64 kDa v-Src-SH3-binding protein, present in detergent-insoluble cellular fractions, was identified as p21ras-GTPase-activating protein-associated p62 (GAPA62). In non-transformed cells, including NIH 3T3 cells, GAPA62 was present in both the RIP A-soluble and RIP A-insoluble fractions, but only the latter form was tyrosine-phosphorylated. In contrast, in polyoma middle T antigen-transformed 3T3 cells, GAPA62 was present only in the RIP A-insoluble fraction, where it was highly phosphorylated. It is suggested that tyrosine phosphorylation of GAPA62 may be an important determinant of its cellular localization and its possible function as a mediator of v-Src actions.

Release of gelatinase A (matrix metalloproteinase 2) induced by photolysis of caged phosphatidic acid in HT 1080 metastatic fibrosarcoma cells.

Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic. We have synthesized a photolyzable derivative of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events. 1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of PA. Photolysis of the compound by ultraviolet light resulted in the formation of phosphatidic acid. The structure of the compound and of its photolytic products was verified by NMR spectroscopy. The utility of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which the formation of PA by phospholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after 120 min of incubation. Ultraviolet illumination of cPA-loaded cells for 5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to the medium, whereas nonphotolyzed cPA caused no change in MMP-2 release. Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly introduced PA. Thus, the effect of laminin on MMP-2 secretion can be mimicked by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis. These results indicate that cPA could serve as a unique tool for studying the cellular roles of PA.