Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function.

In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N-terminal region with transcription factor IIB.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation.

A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction of the E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.

NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.

Study of the role of retinoblastoma protein in terminal differentiation of murine erythroleukemia cells.

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.