Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.     

Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.     

Multiple functional proteins are produced by cleaving Asn-Gln bonds of a single precursor by vacuolar processing enzyme

Precursor-accumulating vesicles mediate transport of the precursors of seed proteins to protein storage vacuoles in maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97,310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CXXXC motifs, a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Both immunocytochemistry and immunoblots with anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa vicilin-like protein were accumulated in the vacuoles. To identify the mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass spectrometry and peptide sequencing showed that two Cys-rich peptides, three Arg/Glu-rich peptides, and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a pyroglutamate at their NH2 termini. To clarify the cleavage mechanism, in vitro processing of PV100 was performed with purified vacuolar processing enzyme (VPE). Taken together, these results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins. It is likely that the Asn-Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. We also found that the Cys-rich peptide functions as a trypsin inhibitor. Our findings suggested that PV100 is converted into different functional proteins, such as a proteinase inhibitor and a storage protein, in the vacuoles of seed cells.     

Roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein

The spike (S) protein of coronavirus mouse hepatitis virus (MHV), mediates attachment and fusion during viral entry and cell-to-cell fusion later in infection. By analogy with other viral proteins that induce cell fusion the MHV S protein would be expected to have a hydrophobic stretch of amino acids that serves as a fusion peptide. Sequence analysis suggests that the S protein falls within the group of fusion proteins having internal rather than N-terminal fusion peptides. Based on the features of known viral fusion peptides, we identified two regions (PEP1 and PEP2) of MHV-A59 S2 as possible fusion peptides. Site-directed mutagenesis and an in viro cell-to-cell fusion assay were used to evaluate the roles of PEP1 and PEP2, as well as a third previously identified putative fusion domain (PEP3) in membrane fusion. Substitution of bulky hydrophobic residues with charged residues within PEP1 affects the fusion activity of the S protein without affecting processing and surface expression. Similar substitutions within PEP2 result in a fusion-negative phenotype; however, these mutant S proteins also exhibit defects in protein processing and surface expression which likely explain the loss of the ability to induce fusion. Thus PEP1 remains a candidate fusion peptide, while PEP2 may play a significant role in the overall structure or oligomerization of the S protein. PEP3 is an unlikely putative fusion peptide since it is not conserved among coronaviruses and nonconservative amino acid substitutions in PEP3 have minimal effects on cell-to-cell fusion.     

Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.     

Domain organization of the 39-kDa receptor-associated protein

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.     

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.     

Expression of the recombinant anchorless N-terminal domain of mouse hepatitis virus (MHV) receptor makes hamster of human cells susceptible to MHV infection

Mouse hepatitis virus (MHV) receptor, the receptor for the murine coronavirus MHV, was expressed in MHV-resistant hamster and human cells as a series of mutant, recombinant glycoproteins with carboxy-terminal deletions lacking the cytoplasmic tail, transmembrane domain, and various amounts of the immunoglobulin constant-region-like domains. The soluble receptor glycoproteins containing the N-terminal virus-binding domain were released into the supernatant medium and inactivated the infectivity of MHV-A59 virions in a concentration-dependent manner. Surprisingly, some of the anchorless glycoproteins were found on the plasma membranes of transfected cells by flow cytometry, and these cells were rendered susceptible to infection with three strains of MHV. Thus, in the cells in which the anchorless, recombinant receptor glycoprotein is synthesized, some of the protein is bound to an unidentified moiety on the plasma membrane, which allows it to serve as a functional virus receptor.     

Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD+ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD+. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purified caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.     

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.     

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59)

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.     

Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival

The bcl-2 gene can potentially encode 26- and 22-kDa proteins that differ only in their carboxyl tails because of an alternative splicing mechanism. The larger of these proteins contains a hydrophobic transmembrane domain within its carboxyl terminus, resides (at least in part) in mitochondrial membranes and has been shown to prolong cell survival by blocking programmed cell death (also termed "apoptosis"). To explore the function of the shorter 22-kDa Bcl-2 protein that lacks a transmembrane domain, DNAs encoding p26-Bcl-2-alpha or p22-Bcl-2-beta were expressed in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D. In contrast to p26-Bcl-2 alpha that markedly prolonged cell survival, p22-Bcl-2-beta did not extend the survival of 32D cells when cultured in the absence of IL-3. Expression in 32D cells of a chimeric DNA that fused portions of the open reading frame common to Bcl-2-alpha and Bcl-2-beta (amino-acids 1-195) with sequences encoding the transmembrane and cytosolic domains of the IL-2 receptor-alpha protein resulted in production of a Bcl-2/IL-2R fusion protein that was capable of prolonging 32D cell survival in the setting of IL-3 withdrawal. Based on fractionation of cells to produce crude heavy membrane, light membrane, nuclei, and cytosolic preparations, much of the p22-Bcl-2-beta protein appeared to reside in the cytosol, whereas Bcl-2-alpha and the Bcl-2/IL-2R chimeric proteins were found exclusively in fractions that also contained the inner mitochondrial membrane protein F1-beta-ATPase. Taken together, these findings demonstrate the importance of membrane association for the function and intracellular targeting of the apoptosis-blocking Bcl-2 protein. Furthermore, despite the strong evolutionary conservation of the carboxyl regions of Bcl-2-alpha proteins observed previously for mammalian and avian species, these data suggest that a heterologous transmembrane domain can be substituted without loss of function.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

The assessment of child sexual abuse allegations: using research to guide clinical decision making

Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo. Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.     

Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.     

Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes

In human T-lymphocytes the Src family protein tyrosine kinase p59(fyn) associates with three phosphoproteins of 43, 55, and 85 kDa (pp43, pp55, and pp85). Employing a GST-Fyn-Src homology 2 (SH2) domain fusion protein pp55 was purified from lysates of Jurkat T-cells. Molecular cloning of the pp55 cDNA reveals that the pp55 gene codes for a so far nondescribed polypeptide of 359 amino acids that comprises a pleckstrin homology domain, a C-terminal SH3 domain, as well as several potential tyrosine phosphorylation sites, among which one fulfills the criteria to bind Src-like SH2 domains with high affinity. Consistent with this observation, pp55 selectively binds to isolated SH2 domains of Lck, Lyn, Src, and Fyn but not to the SH2 domains of ZAP70, Syk, Shc, SLP-76, Grb2, phosphatidylinositol 3-kinase, and c-abl in vitro. Based on these properties the protein was termed SKAP55 (src kinase-associated phosphoprotein of 55 kDa). Northern blot analysis shows that SKAP55 mRNA is preferentially expressed in lymphatic tissues. SKAP55 is detected in resting human T-lymphocytes as a constitutively tyrosine phosphorylated protein that selectively interacts with p59(fyn). These data suggest that SKAP55 represents a novel adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes.