Pin1 Catalyzes Conformational Changes of Thr-187 in p27Kip1 and Mediates Its Stability through a Polyubiquitination Process

The cis-trans peptidylprolyl isomerase Pin1 plays a critical role in regulating a subset of phosphoproteins by catalyzing conformational changes on the phosphorylated Ser/Thr-Pro motifs. The phosphorylation-directed ubiquitination is one of the major mechanisms to regulate the abundance of p27^Kip1. In this study, we demonstrate that Pin1 catalyzes the cis-trans conformational changes of p27^Kip1 and further mediates its stability through the polyubiquitination mechanism. Our results show that the phosphorylated Thr-187-Pro motif in p27^Kip1 is a key Pin1-binding site. In addition, NMR analyses show that this phosphorylated Thr-187-Pro site undergoes conformational change catalyzed by Pin1. Moreover, in Pin1 knock-out mouse embryonic fibroblasts, p27^Kip1 has a shorter lifetime and displays a higher degree of polyubiquitination than in Pin1 wild-type mouse embryonic fibroblasts, suggesting that Pin1 plays a critical role in regulating p27^Kip1 degradation. Additionally, Pin1 dramatically reduces the interaction between p27^Kip1 and Cks1, possibly via isomerizing the cis-trans conformation of p27^Kip1. Our study thus reveals a novel regulatory mechanism for p27^Kip1 stability and sheds new light on the biological function of Pin1 as a general regulator of protein stability.Cellular differentiation and cell cycle inhibition are tightly controlled via sensitive molecular mechanisms. p27^Kip1, a member of the Cip/Kip family, is an essential cell cycle inhibitor that functions largely during the G₀/G₁ phase where it promotes the assembly of the cyclin D1-CDK4 complex and inhibits the kinase activity of the cyclin E-CDK2 complex in the G₁-S phase (1–4). Several review articles have elegantly summarized and discussed the detailed cellular functions of p27^Kip1 (1–6). p27^Kip1 is also a phosphoprotein with multiple Ser/Thr phosphorylation sites, including Ser-10, Ser-178, and Thr-187, followed by a proline residue. Hence, these motifs are potential substrate sites for proline-directed kinases (5, 6). Compared with Ser-178, which has not yet been well studied, the phosphorylation of Ser-10 and Thr-187 has been well characterized to be important for the regulation of p27^Kip1 function. For instance, Ser-10 has been found to be the major phosphorylation site of p27^Kip1 (7) and to play an important role in regulating cell migration (8–10), although the regulation of Ser-10 phosphorylation is still not completely defined (11, 12).In contrast to Ser-10 and Thr-178, Thr-187 is the best characterized phosphorylation site on p27^Kip1 and is known to regulate the complex formation of p27^Kip1-cyclin E-CDK2 (1–2). In addition, it is also widely accepted that Thr-187 plays a crucial role in determining the abundance of mature p27^Kip1 proteins. The phosphorylation of Thr-187 directs p27^Kip1 to an SCF^Skp2 ubiquitin ligase complex (consisting of Skp2-Skp1-Cks1-Cul1-Roc1), which in turn promotes the polyubiquitination and degradation of p27^Kip1 (13, 14). The crystal structure of the Skp1-Skp2-Cks1-p27^Kip1 phosphopeptide complex shows that p27^Kip1 binds both Cks1 and Skp2 and that the C terminus of Skp2 and Cks1 forms the substrate recognition core of the SCF complex (15). Furthermore, the structure of this complex has revealed that the phosphorylation of Thr-187 in p27^Kip1 is recognized by the phosphate-binding site of Cks1, indicating that Cks1 is not only a facilitator but also an indispensable component in p27^Kip1 degradation machinery (15).Pin1 is a unique peptidyl-prolyl isomerase (PPIase)² that recognizes only the phosphorylated Ser/Thr motif preceding a proline residue (16). In addition, Pin1 is very prominent in isomerizing the cis-trans conformation of prolyl-peptidyl bonds in its substrates, resulting in either the modification of their function (e.g. c-Jun (17), β-catenin (18), Bax (19), and Notch1 (20)) or modulation of their stability (e.g. cyclin D1 (21), p53 (22, 23), and NF-κB (24)). Loss of Pin1 in mice results in several phenotypes similar to those of cyclin D1-null mice (21) and neuronal degenerative phenotypes (25–28), suggesting the conformational changes mediated by Pin1 may be crucial for the normal functioning of cells. Additionally, Pin1 also plays important roles in cancer and other cellular events, which have been extensively discussed in several recent review articles (29–33).In this study, we show that Pin1 binds to p27^Kip1, mainly through the phosphorylated Thr-187-Pro motif, and causes subsequent prolyl isomerization of this cell cycle protein. Moreover, we also find that Pin1 can protect p27^Kip1 from degradation. Importantly, we demonstrate that by catalyzing conformational changes in p27^Kip1, Pin1 hinders its association with Cks1, resulting in a reduction of polyubiquitination of p27^Kip1 and protecting its degradation by SCF^Skp2 complexes. Our results suggest that the cis-trans isomerization catalyzed by Pin1 represents a novel regulatory mechanism during post-phosphorylation of proteins and polyubiquitination-directed degradation pathways.     

The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.Dissemination of tumor cells is strictly linked to their ability to attach to and move within the extracellular matrix (ECM) in a three-dimensional (3D) environment. The use of 3D experimental model systems revealed that a higher complexity in cell migration and adaptation responses exists in the 3D model than in the classical 2D model (10, 16, 41, 49). A striking example is given by the fact that only in 3D could individually migrating cells use different mechanisms such as mesenchymal and amoeboid motility (16, 17). The relative slow mesenchymal migration is characterized by a fibroblast-like spindle shape and is dependent on integrin-mediated adhesion and on protease function (16). The amoeboid motility can in some cases represent a less adhesive, integrin-independent type of movement. Cells use a propulsive mechanism and are highly deformable, and rather than degrade the matrix, they are able to squeeze through it (16). As a result, the cells that use the amoeboid motility can potentially move faster than cells that use a mesenchymal strategy. Mesenchymal and amoeboid movements are also characterized by a different involvement of small GTPases of the Rho family. A high RhoA activity is associated mainly with the amoeboid motility, while the mesenchymal migration needs a high Rac activity at the leading edge to promote the extension of cellular protrusions (41, 48). Under certain circumstances, cancer cells can undergo conversion from a mesenchymal toward an amoeboid motility, an event referred as mesenchymal-amoeboid transition (MAT) (50). MAT represents a putative escape mechanism in tumor cell dissemination that could be induced by inhibition of pericellular proteolysis (50) or by increased membrane-associated RhoA activity (18, 40).Key mediators of cell motility through ECM substrates are the members of the Src family kinases. The prototype of Src family kinases, c-Src (14), is activated following cell-ECM adhesion and contributes to regulate the focal adhesion turnover and the cytoskeletal modifications necessary for normal cell adhesion and motility (52). The c-Src gene is the proto-oncogene of the transforming gene v-src of Rous sarcoma virus, and its elevated protein level and activity have been found in many human tumors (20, 28, 27, 34). Despite the accumulation of information and new molecular understanding of how Src is controlled, there is still an incomplete picture about its role in the generation of the malignant phenotype. v-Src shows higher levels of the kinase activity and transforming ability than c-Src (14, 15, 52). It induces normal cells to acquire a variety of transformed features, including alteration of morphology and increase of invasion ability due to its role in focal adhesion remodeling (7, 9, 13).Many data suggest that there is a close relationship between cell-ECM interaction and the proliferation and movements in both normal and tumor cells (5, 38, 43). Accordingly, Src activation may influence not only cell motility but also cell cycle progression by targeting the cell cycle inhibitor p27^kip1 to proteasomal degradation (22, 39). Recent evidences indicated that p27^kip1 (hereafter called p27) can also regulate cell migration, even though its role still remains controversial since it has been reported to either block or stimulate cell movements (1, 4, 11, 19, 21, 23, 29, 45).Based on these notions, we tested the possible contribution of p27 to the growth and motility phenotypes induced by v-src transformation, with special regard to those cellular invasive features that can be observed in 3D environments. By studying in vitro and in vivo the behavior of wild-type (WT) and p27-null fibroblasts transformed with v-src, we highlight a new role for p27 in the regulation of cellular plasticity that can ultimately drive tumor cell shape, motility, and invasion.     

Ubiquitin-Independent Degradation of Antiapoptotic MCL-1

Antiapoptotic myeloid cell leukemia 1 (MCL-1) is an essential modulator of survival during the development and maintenance of a variety of cell lineages. Its turnover, believed to be mediated by the ubiquitin-proteasome system, facilitates apoptosis induction in response to cellular stress. To investigate the contribution of ubiquitinylation in regulating murine MCL-1 turnover, we generated an MCL-1 mutant lacking the lysine residues required for ubiquitinylation (MCL-1^KR). Here, we demonstrate that despite failing to be ubiquitinylated, the MCL-1^KR protein is eliminated at a rate similar to that of wild-type MCL-1 under basal and stressed conditions. Moreover, the degradation of wild-type MCL-1 is not affected when ubiquitin-activating enzyme E1 activity is blocked. Likewise, both wild-type and MCL-1^KR proteins are similarly degraded when expressed in primary lymphocytes. Supporting these findings, unmodified, in vitro-translated MCL-1 can be degraded in a cell-free system by the 20S proteasome. Taken together, these data demonstrate that MCL-1 degradation can occur independently of ubiquitinylation.Apoptosis is a genetically controlled cell suicide program which is regulated by members of the BCL-2 family and is essential for development and maintenance of multicellular organisms (16). Antiapoptotic MCL-1 was identified as a gene induced in a human myeloblastic leukemia cell line treated to differentiate in vitro and found to have homology with antiapoptotic BCL-2 (34, 69). Its germ line ablation results in an early embryonic lethality, and lineage-specific gene ablation has revealed that Mcl-1 is essential for maintaining the survival of lymphocytes, neutrophils, neurons, bone marrow progenitors, and hematopoietic stem cells (4, 25, 50, 51, 54, 58).MCL-1''s short half-life (estimated at less than 1 h) is unique among antiapoptotic BCL-2 family members. Under basal conditions, human MCL-1 undergoes rapid protein turnover, but the control of this constitutive degradation pathway is incompletely understood (48). MCL-1 possesses a distinctive amino terminus and several proline-glutamic acid-serine-threonine (PEST)-rich regions; however, its turnover appears to be unaffected by deletion of these regions (2). MCL-1 can be cleaved by caspases and granzyme B, which proteolytically degrade MCL-1 during cell death (14, 27, 29). In addition, human MCL-1 can be ubiquitinylated and degraded by the proteasome (48). Several levels of degradation control have been postulated. In HeLa cells, MCL-1 undergoes constitutive protein turnover independent of cell death signaling (48). In contrast, in growth factor-dependent cells, regulation of MCL-1 stability is modulated by cytokine signaling (20, 21, 41, 71).Ubiquitinylation of target proteins is processed through a multienzyme cascade (30). Ubiquitin is activated by one of two ubiquitin-activating enzymes (E1), transferred to one of several ubiquitin-conjugating enzymes (E2), and then finally transferred to one of multiple ubiquitin protein ligases (E3), which covalently attaches ubiquitin to lysine residues within the target protein. Polyubiquitin chains, formed by the successive conjugation of ubiquitin to internal lysine residues of the previously conjugated ubiquitin moiety, are recognized and degraded by the 26S proteasome (56).Two E3 ligases have been implicated in mediating MCL-1 ubiquitinylation. MULE (also called LASAU1, ARF-BP1, or HUWE1), a HECT-domain (homologous to the E6-AP carboxyl terminus) family E3 ligase, possesses a BH3 domain similar to that of proapoptotic BAK that allows it to selectively target MCL-1 (65, 72). While MULE may target MCL-1 for degradation, it is not solely MCL-1 specific, as it also ubiquitinylates p53, E3^Histone, c-Myc, and CDC-6 (1, 9, 26, 40). The other E3 ligase, β-TrCP, is a Skp1-CUL1-F box protein (SCF) family member that requires MCL-1 phosphorylation by GSK3 to mediate recognition (21). Like MULE, β-TrCP has other substrate proteins (including BIM, Emi-1, β-catenin, IκB-α, PERIOD2, etc.) (7, 19, 28, 53, 68, 70). Recently, the ubiquitin-specific peptidase 9 X-linked (USP9X) deubiquitinylase (DUB) was identified as a regulator of MCL-1, as RNA interference (RNAi)-mediated silencing of USP9X led to a loss of MCL-1 without affecting its mRNA expression (57). However, USP9X has other substrates, including ASK1, AMPK, Smad4, MARCH1, and Itch (3, 24, 44, 46, 47). Therefore, it is unclear what the relative contributions of the E3 ligases and DUB are in regulating MCL-1 expression, and the multitude of substrates makes it difficult to unravel how this dynamic network is regulated.To investigate the role of ubiquitinylation in modulating murine MCL-1 protein levels, we generated an allele of murine Mcl-1 that lacks the sites for ubiquitinylation (Mcl-1^KR) by mutating all 14 lysine residues to arginine. Surprisingly, MCL-1^KR is turned over essentially identically to wild-type MCL-1 under basal and stressed conditions, suggesting the existence of an alternative degradation pathway. To support these findings, we utilized temperature-sensitive ubiquitin-activating enzyme E1 (UBE1)-expressing cells to abolish ubiquitinylation of endogenous MCL-1. In these cells, MCL-1 protein is degraded at a similar rate when the E1 was either active or inactive, consistent with MCL-1 protein being eliminated in a ubiquitinylation-independent manner. To assess the role of ubiquitinylation in regulating MCL-1 turnover in vivo, we generated transgenic mice that express epitope-tagged versions of wild-type MCL-1 or MCL-1^KR. In lymphocytes derived from these mice, both versions of MCL-1 were eliminated at similar rates, demonstrating that ubiquitinylation is not essential for MCL-1 protein turnover. Lastly, in vitro-translated MCL-1 protein can be degraded in a cell-free system by the 20S proteasome in the absence of ubiquitinylation. Together, these data demonstrate that while MCL-1 can be the target of ubiquitinylation, this is not the only pathway through which its steady-state protein levels are regulated.     

PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development

One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.Polypeptide growth factors, such as insulin, insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang1), exert biological functions through binding to their transmembrane receptors that belong to a large family of receptor tyrosine kinases (RTKs) (4). Consequently, the receptor molecules form homo- or heterodimers, and the intracellular tyrosines at the carboxyl termini of the receptors become phosphorylated (37). Numerous distinct adaptor/regulatory proteins, through their Src homologous 2 (SH2) domains, bind to the phosphotyrosines and transduce the signal to downstream pathways, among which are two essential and well-characterized signaling cascades—the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt signaling pathways (4, 13, 37).The regulatory subunit of PI3K, p85, possesses the SH2 domain and can, therefore, bind to phosphotyrosines on the RTKs and subsequently render activation of the catalytic subunit of PI3K, p110 (7, 8). Active p110 phosphorylates phosphoinositide biphosphate (PIP2), turning it into PIP3 that recruits PDK1 and Akt to the cellular membrane, where Akt is phosphorylated at threonine 308 (T308 for Akt1) by PDK (5, 23, 30). The serine 473 (S473) of Akt (Akt1) is phosphorylated by mTOR complex 2 (mTORC2) and other kinases (17, 36). Phosphorylation of Akt at these two amino acids brings it to full activation. In PDK1-deficient embryonic stem (ES) cells, T308 phosphorylation was abolished and most of the Akt activity was lost, although the S473 phosphorylation was intact (40).Akt plays an important role in multiple biological processes, such as cell survival, growth, glucose metabolism, and angiogenesis (2, 12, 14-16, 22, 23, 39, 41-43). In mammals, there are three Akt isoforms, termed Akt 1, -2, and -3. Previously, we generated Akt1- and Akt3-deficient mice and studied their roles in mouse development (2, 15, 39, 42, 43). We found that the Akt1 and -3 double knockout (KO) (DKO) mice were embryonically lethal at around embryonic day 12 (E12) and manifested developmental defects in multiple tissues, including the cardiovascular system (14, 15, 43). These studies suggest that the Akt signaling pathway is involved in cardiovascular development.Other than Akt isoforms, PDK1 also activates another group of AGC family kinases, such as p70 ribosomal S6 kinase (S6K) (32), serum, and glucocorticoid-induced protein kinase (SGK) (26), p90 ribosomal S6 kinase (RSK) (21), and atypical isoforms of protein kinase C (PKC) (31). Comprehensive and intensive mouse genetic studies performed mainly by Alessi and coworkers have confirmed the regulation of these AGC kinases by PDK1 (3, 9, 10, 27-29, 40).PDK1 knockout mice were severely growth retarded and died at around E9.0, indicating an essential role of PDK1 in development (27). However, its function and downstream targets in cardiovascular development are still elusive. To study this, we deleted PDK1 specifically in endothelial cells through Cre recombinase-mediated excision (25). The results have revealed an essential role of PDK1 in vascular remodeling and integrity and in cardiac development through activation of Akt and its downstream target of Snail.     

Nef-Induced CD4 Endocytosis in Human Immunodeficiency Virus Type 1 Host Cells: Role of p56lck Kinase

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56^lck kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56^lck in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56^lck-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56^lck with cell surface-expressed CD4. Regardless of the presence of p56^lck, the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.Besides proteins that are essential for proper virus processing and assembly, the genomes of primate lentiviruses such as human immunodeficiency virus type 1 (HIV-1) encode auxiliary proteins that modulate viral infectivity. The 27-kDa auxiliary protein Nef is a key element in the progression of primary HIV-1 infection toward AIDS. Cases of patients infected with HIV-1 strains harboring a deletion in the nef gene or a defective nef allele have been reported. Some of these patients exhibit asymptomatic or slow progression toward the disease (6, 17, 37). In vitro, Nef facilitates viral replication and enhances the infectivity of viral particles (13, 47, 69). The mechanisms involved in the Nef-induced increase of viral infectivity remain elusive; however, it is a multifactorial process related to the ability of Nef to alter the trafficking of host cell proteins.Indeed, the most documented effect of Nef during the course of viral infection is its ability to disturb the clathrin-dependent trafficking machinery involved in the transport of transmembrane proteins through endosomal compartments. This leads to the modulation of the level of cell surface expression for some receptors, including CD4, which is the primary receptor of HIV-1 (35) and major histocompatibility complex class I (reviewed in references 22 and 27). The downregulation of CD4, which results in the impairment of the immunological synapse (72) and the downregulation of major histocompatibility complex class I molecules (reviewed in reference 16), is believed to contribute to the escape of HIV-1-infected cells from immunosurveillance. Moreover, the downregulation of CD4 helps avoid superinfection of cells, which would be deleterious to the virus (reviewed in reference 21), and has a direct impact on viral fitness by allowing better incorporation of the functional envelope in viral particles produced from CD4-expressing cells (3, 36, 53).Nef-induced cell surface downregulation of CD4 is efficient in all CD4-expressing cells and depends on the integrity of a di-Leu motif at position 164/165 of the C-terminal flexible loop of HIV-1 Nef (2, 9, 25). This di-Leu motif allows for the interaction with clathrin-associated adaptor protein (AP) complexes that participate in the clathrin-dependent vesicular transport within the endocytic pathway. The AP type 2 (AP-2) complex is localized at the plasma membrane and is essential to the assembly and function of clathrin-coated pits involved in the internalization of receptors from the cell surface (59). The interaction of Nef with AP-2 is well delineated and has been proposed to enhance the targeting of CD4 to clathrin-coated pits and its internalization (10, 12, 26, 32, 39).Helper T lymphocytes are the predominant cell type that expresses CD4; however, CD4 is also present at the surfaces of monocytes and macrophages (70), where its function is yet to be elucidated. Whereas cell surface CD4 is rapidly internalized in myeloid cells, CD4 is stabilized at the plasma membrane in lymphoid cells through its interaction with the Src family protein tyrosine kinase p56^lck. Cys residues located at positions 420/422 in the CD4 cytoplasmic tail are essential to the constitutive association with p56^lck (73). Besides its role in signal transduction, this interaction also correlates with an accumulation of CD4 in lipid rafts and enhanced exclusion of CD4 from clathrin-coated pits (50).In T cells, treatment with phorbol esters such as phorbol 12-myristate 13-acetate (PMA) provokes the phosphorylation of Ser residues found in the cytoplasmic tail of CD4. This correlates with a decreased association of p56^lck with CD4 and the internalization of the receptor (24, 32-34, 41, 45, 48, 52, 56, 61, 66-68). Nef-induced CD4 downregulation is known to be independent of Ser phosphorylation (20) and is therefore governed by mechanisms different from those involved in PMA-induced CD4 downregulation. However, the Leu-based sorting motif in the CD4 cytoplasmic tail is critical for both PMA and Nef-induced CD4 downregulation (2, 5, 24, 31, 56, 60, 68), thus indicating that despite being different, the mechanisms involved in Nef- and PMA-induced CD4 downregulation partially overlap.In the present study, we investigated whether the mechanisms used by Nef to downregulate CD4 are cell type-dependent processes. We looked at the trafficking and steady-state expression of CD4 in the main target cells of HIV-1, CD4-positive T lymphocytes, and cells of the monocyte/macrophage lineage. Our results demonstrate that the presence of p56^lck has a direct impact on the mechanisms used by Nef to downregulate CD4 from the cell surface of T lymphocytes. They also reveal that Nef uses distinct pathways to decrease levels of cell surface expression of CD4 in lymphoid or myeloid target cells of HIV-1.