Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.

Targeted overexpression of protein kinase C β2 isoform in myocardium causes cardiomyopathy

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the β isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCβ isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCβ2 isoform in the myocardium. These mice overexpressed the PKCβ2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCβ-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCβ2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH–VL domain pairs shows a rearrangement of VH CDR3

The 2.0-Å resolution x-ray crystal structure of a novel trimeric antibody fragment, a “triabody,” has been determined. The trimer is made up of polypeptides constructed in a manner identical to that previously described for some “diabodies”: a VL domain directly fused to the C terminus of a VH domain—i.e., without any linker sequence. The trimer has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. For the particular structure reported here, the polypeptide was constructed with a VH domain from one antibody fused to the VL domain from an unrelated antibody giving rise to “combinatorial” Fvs upon formation of the trimer. The structure shows that the exchange of the VL domain from antibody B1-8, a Vλ domain, with the VL domain from antibody NQ11, a Vκ domain, leads to a dramatic conformational change in the VH CDR3 loop of antibody B1-8. The magnitude of this change is similar to the largest of the conformational changes observed in antibody fragments in response to antigen binding. Combinatorial pairing of VH and VL domains constitutes a major component of antibody diversity. Conformationally flexible antigen-binding sites capable of adapting to the specific CDR3 loop context created upon VH–VL pairing may be employed by the immune system to maximize the structural diversity of the immune response.

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A’s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.

The solution structure of the N-terminal domain of α2-macroglobulin receptor-associated protein

The three-dimensional structure of the N-terminal domain (residues 18–112) of α2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23–34, 39–65, and 73–88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix–loop–helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of ≈20-kDa C-terminal fragments (CTF) and ≈30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181–190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the ≈20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181–190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the α2β1 integrin ligand site DGE. In VP7, the αxβ2 integrin ligand site GPR and the α4β1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express α2β1 and β2 integrins. Rotavirus VP4-mediated cell entry may involve the α2β1 integrin, whereas VP7 appears to interact with αxβ2 and α4β1 integrins.

Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity

Transforming growth factor β (TGF-β)-mediated G1 arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-β-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-β-treated cells failed to induce p15INK4b, down-regulate CDC25A, or increase levels of p21CIP1, p27KIP1, and p57KIP2. However, TGF-β treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr160 phosphorylation on Cdk2. Whole-cell lysates from TGF-β-treated cells showed inhibition of Cdk2 Thr160 Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-β treatment. Taken together, these observations demonstrate that TGF-β signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

Estradiol repression of tumor necrosis factor-α transcription requires estrogen receptor activation function-2 and is enhanced by coactivators

The tumor necrosis factor-α (TNF-α) promoter was used to explore the molecular mechanisms of estradiol (E2)-dependent repression of gene transcription. E2 inhibited basal activity and abolished TNF-α activation of the TNF-α promoter. The E2-inhibitory element was mapped to the −125 to −82 region of the TNF-α promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) β is more potent than ERα at repressing the −1044 TNF-α promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERβ, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E2-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.