A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins.
In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.
Gross
S D
SD
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Hoffman
D P
DP
Fisette
P L
PL
Baas
P
P
Anderson
R A
RA
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D047388:Casein Kinases; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000166:cytology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure; D018377:Neurotransmitter Agents / Q000378:metabolism; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013552:Swine; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism*
D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C489618:Sv2a protein, rat; D011494:Protein Kinases; D047388:Casein Kinases