Simultaneous visualization of the translocation of protein kinase Calpha-green fluorescent protein hybrids and intracellular calcium concentrations
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Simultaneous visualization of the translocation of protein kinase Calpha-green fluorescent protein hybrids and intracellular calcium concentrations. / Almholt, K; Arkhammar, P O; Thastrup, Ole; Tullin, S.
I: Biochemical Journal, Bind 337 ( Pt 2), 1999, s. 211-8.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Simultaneous visualization of the translocation of protein kinase Calpha-green fluorescent protein hybrids and intracellular calcium concentrations
AU - Almholt, K
AU - Arkhammar, P O
AU - Thastrup, Ole
AU - Tullin, S
PY - 1999
Y1 - 1999
N2 - The alpha isoform of protein kinase C (PKCalpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCalpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCalpha in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCalpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCalpha. In contrast, PMA induced translocation of PKCalpha without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCalpha, indicating that PKCalpha translocation may be independent of both actin filaments and microtubules. The time course of PKCalpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.
AB - The alpha isoform of protein kinase C (PKCalpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCalpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCalpha in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCalpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCalpha. In contrast, PMA induced translocation of PKCalpha without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCalpha, indicating that PKCalpha translocation may be independent of both actin filaments and microtubules. The time course of PKCalpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.
KW - Animals
KW - Biological Transport
KW - Calcium
KW - Carbachol
KW - Cytoskeleton
KW - Green Fluorescent Proteins
KW - Guinea Pigs
KW - Image Processing, Computer-Assisted
KW - Isoenzymes
KW - Luminescent Proteins
KW - Mice
KW - Microscopy, Fluorescence
KW - Protein Kinase C
KW - Protein Kinase C-alpha
KW - Recombinant Fusion Proteins
KW - Tetradecanoylphorbol Acetate
M3 - Journal article
C2 - 9882617
VL - 337 ( Pt 2)
SP - 211
EP - 218
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
ER -
ID: 43349181