Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.
Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.
Cressman
C M
CM
Department of Biological Sciences, University of Massachusetts at Lowell 01854, USA.
Shea
T B
TB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008845:Microinjections; D009410:Nerve Degeneration / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000008:administration & dosage / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016875:tau Proteins / Q000378:metabolism*
C056167:Alzheimer's disease antigen; D000911:Antibodies, Monoclonal; D000941:Antigens; D009419:Nerve Tissue Proteins; C085349:PHF-1 monoclonal antibody; D016875:tau Proteins; D011493:Protein Kinase C