scholarly journals Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells

1989 ◽  
Vol 263 (1) ◽  
pp. 223-229 ◽  
Author(s):  
K C Cawley ◽  
C G Akita ◽  
D A Walsh
Keyword(s):  
Kinase Activity ◽  
Phosphorylase Kinase ◽  
3T3 Cells ◽  
Gamma Delta ◽  
Gamma Subunit ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Muscle Phosphorylase ◽  
Nih 3T3 ◽  
Delta Subunit

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.

Regulation of the junB gene by v-src

1992 ◽  
Vol 12 (8) ◽  
pp. 3356-3364
Author(s):  
I Apel ◽  
C L Yu ◽  
T Wang ◽  
C Dobry ◽  
M E Van Antwerp ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Gene Products ◽  
3T3 Cells ◽  
Tyrosine Kinase Activity ◽  
Basal Expression ◽  
High Level ◽  
Transformed Cell Lines ◽  
Point Mutagenesis ◽  
Nih 3T3 ◽  
Nucleotide Region

The proteins encoded by cellular and viral src genes are believed to be involved in the transmission of mitogenic signals, the nuclear recipients of which are largely unknown. In this work, we report that four different v-src-transformed cell lines from three different species possess elevated levels of junB transcripts. Transient expression of junB promoter-chloramphenicol acetyltransferase constructs in NIH 3T3 cells was used to demonstrate that the increase in junB transcripts was specifically associated with v-src expression and could not be recapitulated with a c-src, v-H-ras, or v-raf expression vector. Deletion mutants were used to localize the v-src-responsive region in the junB promoter to a 121-nucleotide region encompassing the CCAAT and TATAA elements. This region is distinct from one in the 5' untranslated region of the junB gene which is required to maintain its high-level basal expression. Point mutagenesis of the junB TATAA box completely abolished v-src responsiveness, suggesting that proteins which bind to this element are modified by src transformation. Several v-src and c-src mutants were used to demonstrate that elevated tyrosine kinase activity of src proteins is required for the observed effects on junB expression. Finally, homology between the TATAA box regions of junB and the unrelated but src-responsive gene 9E3/CEF-4 suggests that modulation of gene activity through proteins which bind to this region may be a recurrent, although not exclusive, theme in src transforming action. Our results suggest that src proteins may modulate some nuclear effectors through pathways not involving cellular ras or raf gene products.

Nuclear localization of phosphatidylinositol 4-kinase β

2002 ◽  
Vol 115 (8) ◽  
pp. 1769-1775 ◽  
Author(s):  
Petra de Graaf ◽  
Elsa E. Klapisz ◽  
Thomas K. F. Schulz ◽  
Alfons F. M. Cremers ◽  
Arie J. Verkleij ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Kinase Activity ◽  
Insoluble Fraction ◽  
Type Ii ◽  
3T3 Cells ◽  
Type Iii ◽  
Pore Complexes ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase,whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,suggesting a specific function for this kinase in nuclear processes.

scholarly journals The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin

1990 ◽  
Vol 268 (2) ◽  
pp. 393-399 ◽  
Author(s):  
H K Paudel ◽  
G M Carlson
Keyword(s):  
Quaternary Structure ◽  
Gel Filtration ◽  
Phosphorylase Kinase ◽  
Gamma Delta ◽  
Gel Filtration Chromatography ◽  
Structural Role ◽  
Low Concentrations ◽  
Gamma Subunits ◽  
Alpha Beta ◽  
Delta Subunit

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, β- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, β- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only β-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

Analysis by mutagenesis of the ATP binding site of the .gamma. subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system

Biochemistry ◽  
1992 ◽  
Vol 31 (43) ◽  
pp. 10616-10625 ◽  
Author(s):  
Jung Hwa Lee ◽  
Susumu Maeda ◽  
Karen L. Angelos ◽  
Shizuo G. Kamita ◽  
Chidambaram Ramachandran ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Binding Site ◽  
Atp Binding ◽  
Phosphorylase Kinase ◽  
Gamma Subunit ◽  
Atp Binding Site ◽  
Baculovirus System ◽  
Muscle Phosphorylase

scholarly journals Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells.

1990 ◽  
Vol 110 (5) ◽  
pp. 1541-1548 ◽  
Author(s):  
A M Honegger ◽  
A Schmidt ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Cell Surface ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Mutant Receptors ◽  
Nih 3T3 ◽  
Type Receptor ◽  
Negative Mutant ◽  
Endocytic Pathways

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.

Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5570-5574
Author(s):  
O Segatto ◽  
C R King ◽  
J H Pierce ◽  
P P Di Fiore ◽  
S A Aaronson
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Transmembrane Domain ◽  
3T3 Cells ◽  
Tyrosine Kinase Activity ◽  
Oncogenic Potential ◽  
Mutant Proteins ◽  
Nih 3T3 ◽  
Abnormal Level

Compared with normal erbB-2 gp185, mutant erbB-2 proteins generated by mutations either in the transmembrane domain or by NH2-terminal deletion are able to transform NIH 3T3 cells at a 10- to 100-fold greater efficiency. Mutant proteins of both classes show increased tyrosine kinase activity, suggesting that an abnormal level of receptor-associated tyrosine kinase activity is a major determinant of erbB-2 oncogenic potential.

scholarly journals Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

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