A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor.

Human immunodeficiency virus 1 (HIV-1) Nef downregulates surface expression of CD4, an integral component of the functional HIV receptor complex, through accelerated endocytosis of surface receptors and diminished transport of CD4 from the Golgi network to the plasma membrane. HIV-1 Nef also diminishes surface expression of major histocompatibility complex (MHC) class I antigens. In the case of HIV-2 and simian immunodeficiency virus 1 (SIV-1) Nef, aminoterminal tyrosine-based motifs mediate the binding of Nef to the AP-1 and AP-2 adaptors and this interaction appears to be required for CD4 downregulation. As these tyrosine motifs are not present in the HIV-1 Nef protein, the molecular basis for the presumed interaction of Nef with components of the endocytic machinery is unknown. Here, we identify a highly conserved dileucine motif in HIV-1 Nef that is required for downregulation of CD4. This motif acts as an internalization signal in the context of a CD8-Nef chimera or in a fusion of the interleukin-2 receptor alpha with an 11-amino-acid region from Nef containing the dileucine motif. Finally, HIV-1 Nef binds to the AP-1 adaptor, both in vitro and in vivo, in a dileucine-dependent manner. We conclude that this conserved dileucine motif in HIV-1 Nef serves as a key interface for interaction with components of the host protein trafficking machinery. Our findings also reveal an evolutionary difference between HIV-1 and HIV-2/SIV in which the Nef proteins utilize structurally distinct motifs for binding cellular adaptors.

Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: analysis of the turnover and distribution of this complex.

The transforming protein of Rous sarcoma virus (RSV), pp60src, was previously shown to associate with two cellular proteins of Mr 90,000 and 50,000 in RSV-transformed chicken cells. In this report, we demonstrate that this interaction is specific for a discrete population of pp60src molecules. Newly synthesized pp60src was found to preferentially associate with pp90 and pp50 to form a short-lived complex. The half-life of this complex varied from 9 to 15 min in cells transformed by nondefective strains of RSV. This interaction between pp60src, pp50, and pp90 took place in a soluble fraction of the cell, and the complex-bound pp60src molecules were not phosphorylated on tyrosine. These results suggest that pp90 and pp50 may be involved in the processing of pp60src molecules before the association of pp60src with the plasma membrane. The kinetics of dissociation of this complex were shown to be altered in cells infected with viruses containing a temperature-sensitive defect in the src gene. When cells infected with these viruses were grown at the nonpermissive temperature, more than 90% of the pp60src molecules were associated with pp90 and pp50, and little or no dissociation was observed in a 3-h chase period. These results suggest that mutations in the src gene which affect the transforming activity of pp60src also affect the stability of the interaction of pp60src with pp90 and pp50.

Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor.

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.

Mammalian cAMP-dependent protein kinase functionally replaces its homolog in yeast.

The cDNA encoding the catalytic subunit (C alpha) from mouse cAMP-dependent protein kinase (PK) was expressed in Saccharomyces cerevisiae. By a plasmid swap procedure, we demonstrated that the mammalian C alpha subunit can functionally replace its yeast homolog to maintain the viability of a yeast strain containing genetic disruptions of the three TPK genes encoding the yeast C subunits. C alpha subunit produced in yeast was purified and its biochemical properties were determined. The protein isolated from yeast appears to be myristylated, as has been found for C subunits from higher eukaryotic cells. This system would be useful for studying the biochemistry of the mammalian enzyme in vitro and its biological role in a model in vivo system. These studies demonstrate that the PK substrate(s) required for viability are recognized by the mammalian enzyme. In general terms, these results demonstrate that heterologous proteins with only 50% sequence conservation with their yeast counterparts can be functional in yeast. This is an important result because it validates the use of yeast to identify the biological role of newly cloned genes from heterologous systems, a key tenet of the Human Genome Initiative.

Functional expression of mammalian adenosine cyclic monophosphate-dependent protein kinase in Saccharomyces cerevisiae.

The heterologous expression of protein kinases in E. coli has proved difficult and unpredictable. Although the v-abl protein kinase is successfully expressed in E. coli, our experiments on expression of yeast C subunits in E. coli produced large amounts of predominantly insoluble and inactive protein. Attempts to refold the protein proved unsuccessful. In contrast, a major fraction of mouse C alpha expressed in E. coli is soluble and the enzyme in the soluble fraction is active; however, certain mutant forms have proved to be unstable, difficult to purify, or insoluble. In addition, the E. coli system cannot be used to study the biological role of posttranslational modifications specific to eukaryotic systems. Several protein kinases have been expressed in soluble form in insect cells using baculovirus, suggesting that this system is generally more reliable than E. coli. However, the presence and nature of posttranslational modifications in insect cells may be different from that found in the natural source and may affect the biochemical function. In addition, baculovirus expression is not particularly useful for studying biological questions. Mouse C alpha and C beta have been overexpressed in NIH3T3 cells. This approach is useful in characterizing the biochemical properties of C alpha versus C beta, but it may not be an ideal system for studying mutant proteins since wild-type C subunits are still expressed from the chromosomal copies in this genetic background. This small level of wild type may make it difficult to analyze weakly functional mutants, which have activities less than 10% that of wild type. Several cell lines with altered subunits of cAMP-dependent protein kinase have been identified but a strain completely devoid of C subunit has not been adequately characterized for protein structure/function studies. Disruption of the genes encoding cAMP-dependent protein kinase in mammalian cells has not yet been accomplished. This chapter describes a method to express a C subunit of mammalian cAMP-dependent kinase in yeast. We have demonstrated that the mouse C alpha subunit can substitute for its yeast counterpart. Since at least one functional C subunit is required for viability, these results suggest that the yeast substrates important for viability are recognized by the mammalian C subunit. Although the sequence conservation between yeast and mouse C subunit is only about 50%, these results demonstrate that heterologous proteins with relatively low sequence conservation with their yeast counterparts can be functional in yeast.(ABSTRACT TRUNCATED AT 400 WORDS)

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.

cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes.

cAPK has provided many insights into the functioning of the diverse family of eukaryotic protein kinases. The fact that a particular amino acid in the catalytic core is conserved is an indication that the residue plays an important role; however, questions concerning function remain obscure. With the catalytic subunit, the assignment of amino acids that participate in catalysis has begun, and in many instances that function appears to be conserved in the other protein kinases. Although the regulatory subunit and the use of cAMP to release its inhibitor effects is unique to cAPK, the general mechanism of a small autoinhibitory region occupying the peptide binding site and thus preventing access of other substrates may be invoked frequently by other protein kinases. Coupling recombinant approaches with protein chemistry is allowing us to decipher at least some of the molecular events associated with cAMP-binding and holoenzyme activation. Although the next chapter in the history of cAPK will undoubtedly include three-dimensional structures, the chemical information remains as an essential complement for interpreting those structures and eventually understanding the molecular events associated with catalysis and activation.

N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability.

Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.

Investigations of the expression of the cellular src gene product.

We have examined the expression of the c-src gene product in a variety of embryonic and adult tissues, in peripheral blood cells, and in cells transformed by other tumor viruses, in an attempt to identify the types of cells in which pp60c-src might provide a specific function. These studies have indicated that c-src gene expression is regulated at multiple levels in different cell types and have suggested that pp60c-src is not exclusively involved in the regulation of cell proliferation. The lowest levels of pp60c-src were found in fibroblasts, which have previously served as the standard cell type for comparisons between pp60c-src and pp60v-src. The highest levels of pp60c-src-specific kinase activity were detected in three types of cells: neurons, platelets, and polyoma virus transformed cells. In this report, we will compare the expression of pp60c-src in fibroblasts to that in platelets, neurons, and polyoma virus transformed fibroblasts. In each of the three latter cell types, the c-src gene product displays a unique pattern of expression which can be distinguished from that in fibroblasts (see diagram Fig. 1).

DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase.

We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.

Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen.

We have examined the in vitro phosphorylation of cellular src protein (pp60c-src) molecules associated with the polyoma virus middle-sized tumor antigen in polyoma virus-transformed cells. These pp60c-src molecules possessed an enhanced tyrosyl kinase activity, migrated aberrantly on NaDodSO4/polyacrylamide gels, and contained a novel site of tyrosine phosphorylation within the amino-terminal region of the molecule. The pp60c-src molecules not associated with the middle-sized tumor antigen were phosphorylated exclusively on a tyrosine residue within the carboxyl-terminal domain of pp60c-src. A similar modified form of the middle-sized tumor antigen-associated pp60c-src protein was detected in lysates from polyoma virus-transformed cells labeled in vivo with [32P]orthophosphate in the presence of sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatases.

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli.

When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.

Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E. coli is due to autophosphorylation. Whether this process is intramolecular or intermolecular cannot be distinguished. Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.

A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src.

Previous studies have shown that the avian progesterone receptor, when in the nontransformed 8 S state, is complexed to another cellular protein having a molecular weight of 90,000. In this report, we show that this receptor-binding protein is indistinguishable from the 90,000-dalton protein which associates in a complex with the Rous sarcoma virus transforming protein, pp60v-src. This identity was established by the following criteria. 1) Monoclonal antibodies directed against the pp60v-src-associated 90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay. Conversely, monoclonal antibodies that recognize the progesterone receptor binding protein bind to the 90-kDa protein which complexes with pp60v-src. 2) Peptide maps prepared from the 90-kDa proteins immunoprecipitated from chicken cells with monoclonal antibodies directed against either the 90-kDa receptor binding protein or the 90-kDa pp60v-src-associated protein were indistinguishable. 3) Preincubation of the progesterone receptor complex with monoclonal antibodies prepared against the pp60v-src-associated protein caused a shift in the sedimentation of the progesterone receptor. Previous studies have established that the pp60v-src-associated protein is indistinguishable from one of the major heat shock proteins which are induced under a variety of stress conditions in eukaryotic cells. These present studies implicate a new role for this 90-kDa protein in the action of steroid hormones.

Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation.

We examine the interaction between polyoma-virus-encoded middle tumor antigen and the cellular src gene product, pp60c-src, using a series of monoclonal antibodies that recognize mammalian pp60c-src. Our results show that infection of mouse cells with transformation-competent strains of polyoma virus results in the stimulation of pp60c-src kinase activity severalfold over that observed in uninfected mouse cells and mouse cells infected with transformation-deficient polyoma virus. A similar degree of enhancement of pp60c-src kinase activity was found in polyoma-virus-transformed rodent cells. No differences were detected in the level of pp60c-src synthesis in polyoma-virus-infected and uninfected mouse cells or polyoma-virus-transformed and normal rodent cells. These studies demonstrate that polyoma-virus-encoded middle tumor antigen is associated with pp60c-src in lysates of polyoma-virus-infected and polyoma-virus-transformed cells and suggest a novel mechanism for the functional activation of a cellular proto-oncogene product, namely, that the interaction between middle tumor antigen and pp60c-src leads to a stimulation of pp60c-src tyrosyl kinase activity.

Diminished spontaneous apoptosis in lymphocytes from human immunodeficiency virus-infected long-term nonprogressors.

The relationship between peripheral lymphocyte apoptosis and human immunodeficiency virus disease progression was studied in infected subgroups with distinct profiles of progression. Long-term nonprogressors (LTNP) and seronegative controls had levels of spontaneous apoptosis significantly lower than those for recent seroconverters who had CD4 cell counts similar to those of nonprogressors but with a high likelihood of disease progression. Lymphocytes from nonprogressors and seronegative controls also showed negligible spontaneous caspase-3 activity, a biochemical indicator for apoptosis, whereas early progressors exhibited substantial activity. In contrast, when activated with mitogens, the lymphocytes from both LTNP and progressors displayed indistinguishable levels of heightened apoptosis. Spontaneous apoptosis and plasma viremia levels correlated positively in progressors, but not in LTNP. These findings demonstrate that increased lymphocyte apoptosis is evident prior to CD4 T cell decline and that LTNP are relatively resistant to the factors that induce accentuated levels of spontaneous but not mitogen-induced cell death.

Crystal structures of the catalytic subunit of cAMP-dependent protein kinase reveal general features of the protein kinase family.

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase serves as a template for the catalytic core of all eukaryotic protein kinases. The various crystal structures are reviewed with particular emphasis on the numerous conserved residues that converge at the active site. The structures also reveal the importance of posttranslational modifications, including myristylation and phosphorylation.

cAMP-dependent protein kinase defines a family of enzymes.

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E. coli, the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thr197 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

Characterization of the altered form of the c-src gene product in neuronal cells.

The pp60c-src protein that is expressed at high levels in cultures of neurons from rat embryos displays an altered mobility on SDS-polyacrylamide gels due to a structural difference in the amino-terminal region of the molecule. In this report we show that the expression of this unique form of pp60c-src, designated pp60c-src(+), is not restricted to cultured neuronal cells since the pp60c-src molecules expressed in tissues from avian and rat neural tissues also display a retarded electrophoretic mobility. The amino-terminal region from pp60c-src(+) was found to contain a novel phosphorylated tryptic peptide that contains phosphoserine. However, this phosphorylation does not appear to be responsible for the retarded electrophoretic mobility of pp60c-src(+), since the mobility of this protein is not altered by phosphatase treatment under conditions that remove greater than 95% of the radiolabeled phosphate on pp60c-src(+). The altered electrophoretic form of pp60c-src was also shown to be radiolabeled with [3H]myristate, indicating that pp60c-src is fatty-acylated in neurons, as is pp60c-src in fibroblasts. The pp60c-src molecules synthesized in vitro using rabbit reticulocyte lysates programmed with mRNA from embryonic brain migrated more slowly on SDS-polyacrylamide gels than the pp60c-src protein that was translated in vitro using RNA from embryonic limb tissue. These results suggest the possibility that the c-src mRNA expressed in neurons may undergo a unique form of processing to generate the structurally distinct form of neuronal pp60c-src(+).