Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 91-102 ◽  
Author(s):  
Y. Grinblat ◽  
S. Zusman ◽  
G. Yee ◽  
R.O. Hynes ◽  
F.C. Kafatos
Keyword(s):  
Germ Line ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
P Element ◽  
Integrin Subunit ◽  
Wild Type ◽  
Mutant Proteins ◽  
Adhesive Interactions

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

1999 ◽  
Vol 144 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Rachel Hellman ◽  
Marc Vanhove ◽  
Annabelle Lejeune ◽  
Fred J. Stevens ◽  
Linda M. Hendershot
Keyword(s):  
Secretory Pathway ◽  
Variable Region ◽  
Wild Type ◽  
Mutant Proteins ◽  
Protein Subunits ◽  
Hsp70 Family ◽  
Nascent Chain ◽  
Kinetic Traps

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

scholarly journals Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

1993 ◽  
Vol 292 (3) ◽  
pp. 833-838 ◽  
Author(s):  
O Rosorius ◽  
G Mieskes ◽  
O G Issinger ◽  
C Körner ◽  
B Schmidt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Phosphorylation Sites ◽  
Calmodulin Kinase ◽  
Major Species ◽  
Mannose 6 Phosphate ◽  
Inhibition Studies

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.

scholarly journals The SR protein B52/SRp55 is essential for Drosophila development.

1994 ◽  
Vol 14 (11) ◽  
pp. 7499-7506 ◽  
Author(s):  
H Z Ring ◽  
J T Lis
Keyword(s):  
Germ Line ◽  
Essential Gene ◽  
Critical Role ◽  
Protein Family ◽  
P Element ◽  
Null Mutant ◽  
Sr Protein ◽  
Drosophila Development

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates

2000 ◽  
Vol 74 (14) ◽  
pp. 6448-6458 ◽  
Author(s):  
Tao Tao ◽  
Mario H. Skiadopoulos ◽  
Fatemeh Davoodi ◽  
Jeffrey M. Riggs ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virus Type ◽  
Vaccine Candidate ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Parainfluenza Virus ◽  
Tail Domain ◽  
Wild Type ◽  
Vaccine Candidates

ABSTRACT We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuatedcp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

The SR protein B52/SRp55 is essential for Drosophila development

1994 ◽  
Vol 14 (11) ◽  
pp. 7499-7506
Author(s):  
H Z Ring ◽  
J T Lis
Keyword(s):  
Germ Line ◽  
Essential Gene ◽  
Critical Role ◽  
Protein Family ◽  
P Element ◽  
Null Mutant ◽  
Sr Protein ◽  
Drosophila Development

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.

scholarly journals Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo.

1995 ◽  
Vol 130 (2) ◽  
pp. 441-450 ◽  
Author(s):  
E J Filardo ◽  
P C Brooks ◽  
S L Deming ◽  
C Damsky ◽  
D A Cheresh
Keyword(s):  
Cell Migration ◽  
Cytoplasmic Tail ◽  
Melanoma Cells ◽  
Structural Motif ◽  
Wild Type ◽  
Npxy Motif ◽  
Alpha V Beta 3 ◽  
Integrin Beta 3

The NPXY sequence is highly conserved among integrin beta subunit cytoplasmic tails, suggesting that it plays a fundamental role in regulating integrin-mediated function. Evidence is provided that the NPXY structural motif within the beta 3 subunit, comprising residues 744-747, is essential for cell morphological and migratory responses mediated by integrin alpha v beta 3 in vitro and in vivo. Transfection of CS-1 melanoma cells with a cDNA encoding the wild-type integrin beta 3 subunit, results in de novo alpha v beta 3 expression and cell attachment, spreading, and migration on vitronectin. CS-1 cells expressing alpha v beta 3 with mutations that disrupt the NPXY sequence interact with soluble vitronectin or an RGD peptide, yet fail to attach, spread, or migrate on immobilized ligand. The biological consequences of these observations are underscored by the finding that CS-1 cells expressing wild-type alpha v beta 3 acquire the capacity to form spontaneous pulmonary metastases in the chick embryo when grown on the chorioallantoic membrane. However, migration-deficient CS-1 cells expressing alpha v beta 3 with mutations in the NPXY sequence lose this ability to metastasize. These findings demonstrate that the NPXY motif within the integrin beta 3 cytoplasmic tail is essential for alpha v beta 3-dependent post-ligand binding events involved in cell migration and the metastatic phenotype of melanoma cells.

scholarly journals Transmembrane glycoprotein gp150 is a substrate for receptor tyrosine phosphatase DPTP10D in Drosophila cells.

1997 ◽  
Vol 17 (12) ◽  
pp. 6859-6867 ◽  
Author(s):  
S J Fashena ◽  
K Zinn
Keyword(s):  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cytoplasmic Domain ◽  
Receptor Protein ◽  
S2 Cells ◽  
Wild Type ◽  
Downstream Signaling ◽  
Transmembrane Glycoprotein

We have begun to explore the downstream signaling pathways of receptor protein tyrosine phosphatases (RPTPs) that control axon guidance decisions in the Drosophila central nervous system. We have focused our studies on the adhesion molecule-like gp150 protein, which binds directly to and is an in vitro substrate for the RPTP DPTP10D. Here we show that gp150 and DPTP10D form stable complexes in Drosophila Schneider 2 (S2) cells and in wild-type larval tissue. We also demonstrate that the DPTP10D cytoplasmic domain is sufficient to confer binding to gp150. gp150 has a short cytoplasmic domain containing four tyrosines, all found within sequences similar to immunoreceptor family tyrosine-based activation motifs (ITAMs). We demonstrate that gp150 is tyrosine phosphorylated in wild-type larvae. In S2 cells, gp150 becomes tyrosine phosphorylated following incubation with PTP inhibitors or upon coexpression of the Dsrc tyrosine kinase. Phosphorylated Dsrc and an unknown 40-kDa phosphoprotein form stable complexes with gp150, thereby implicating them in a putative gp150 signaling pathway. When coexpressed with gp150, either full-length DPTP10D or its cytoplasmic domain mediates gp150 dephosphorylation whereas a catalytically inactive DPTP10D cytoplasmic domain does not. The neural RPTP DPTP99A can also induce gp150 dephosphorylation but does not coimmunoprecipitate with gp150. Taken together, the results suggest that gp150 transduces signals via phosphorylation of its ITAM-like elements. Phosphotyrosines on gp150 might function as binding sites for downstream signaling molecules, thereby initiating a signaling cascade that could be modulated in vivo by RPTPs such as DPTP10D.

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