Evidence that the major postsynaptic density protein is a component of a Ca2+/calmodulin-dependent protein kinase.

Abstract

Polypeptides of Mr 50,000 and 60,000 in isolated synaptic junctions have been compared to polypeptides of corresponding molecular weight in Ca2+/calmodulin-dependent protein kinase II. The polypeptides of corresponding molecular weight from the two preparations were shown by several criteria to be indistinguishable. These criteria included 12'I-labeled tryptic/chymotryptic peptide patterns, 32P-labeled proteolytic peptide maps, and crossreactivity on immunoblots using polyclonal and monoclonal antibodies. Furthermore, studies examining the phosphorylation of substrate proteins, by the endogenous synaptic junction kinase and by Ca2+/calmodulin-dependent protein kinase II, indicated that the two enzymes have similar substrate specificities. Since the Mr 50,000 polypeptide present in synaptic junctions is known to be the major postsynaptic density protein, the present results indicate that the major postsynaptic density protein is a component of Ca2+/calmodulin-dependent protein kinase II.A number of Ca2+/calmodulin-dependent protein kinases have recently been identified in neuronal tissue (for review, see ref. 1). One of these is a kinase purified from rat brain (2) that phosphorylates synapsin I, a synaptic vesicle-associated neuronal protein (3,4). This kinase, designated Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II), has recently been purified to near homogeneity (5, 6) and exhibits a major Mr 50,000 polypeptide and a less prominent Mr 60,000 polypeptide when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. These Mr 50,000 and 60,000 polypeptides coelute with the peak of kinase activity through a variety of purification procedures, are themselves phosphorylated in a Ca2+/calmodulin-dependent manner, and are calmodulin-binding proteins (5,6).Grab et al. (7) found a calmodulin-sensitive protein kinase in a postsynaptic density (PSD) preparation isolated from canine cerebral cortex. An endogenous Mr 50,000 polypeptide is both a substrate for this kinase (7) and a calmodulin-binding protein (8). This Mr 50,000 polypeptide is the most abundant PSD protein and has been designated the major PSD protein (mPSDp) (9-11). This PSD preparation also contains a substrate protein (7) and a calmodulin-binding protein (8) in the Mr 60,000 range. Substrate proteins (12) and calmodulinbinding proteins (13,14) of Mr 50,000 and 60,000 have also been observed in PSD and synaptic junction (SJ) preparations isolated from rat forebrain. The SJ Mr 50,000 polypeptide is identical to the mPSDp (11,14). Therefore, either SJ or PSD preparations can be used as a source for the mPSDp.The similarities between the Mr 50,000 and 60,000 polypeptides in calmodulin kinase II and those of corresponding molecular weight in SJ preparations prompted us to examine their possible relationship. A preliminary account of this work has been reported (15). METHODSSubcellular Fractionation. Whole forebrains were used to isolate SJ fractions by the iodonitrotetrazolium violet/Triton X-100 method as described (11). Care was t...

Differential Inactivation of Postsynaptic Density‐Associated and Soluble Ca2+/Calmodulin‐Dependent Protein Kinase II by Protein Phosphatases 1 and 2A

“…Because targeting of CaMKII to PSDs appears to regulate its availability to protein phosphatases, an important question then becomes what proportion of CaMKII is associated with PSDs in living neurons. Although CaMKII is frequently cited as a major PSD protein, constituting as much as 50% of total protein in isolated PSDs (Kennedy et al, 1983;Goldenring et al, 1984;Kelly et al, 1984), more recent data indicate that much of the CaMKII associated with isolated PSDs can result from rapid postmortem translocation to the PSD before tissue homogenization (Suzuki et al, 1994). When PSDs are isolated from very rapidly homogenized brains, CaMKII is only two-to threefold enriched in PSDs (Suzuki et al, 1994) over whole forebrain extracts (Erondu and Kennedy, 1985).…”

Calcium/Calmodulin-Dependent Protein Kinase II

“…Within the neuron, CaMkinase II appears to be distributed in the spines, somata, axons, dendrites and nerve terminal, with little in the nuclei [38]. In the dendrites of forebrain the kinase is particularly concentrated in the postsynaptic density, where the a subunit is the major postsynaptic density protein of 50 kDa, constituting 30 50 o of the total protein [40][41][42]. Subunit structure As noted above, CaM-kinase II is a heteropolymer comprised of subunits in the range 50-62 kDa depending on the tissue and species.…”

“…In this context, the postsynaptic density is of particular interest since approx. 30 50%o of postsynaptic density protein is the a-subunit of CaM-kinase II [40][41][42].…”

Activity-Dependent Regulation of Calcium/Calmodulin-Dependent Protein Kinase II Localization: Figure 1.

“…In brain, a significant fraction of the enzyme can be readily extracted in a soluble form, and the remainder is associated with the cytoskeleton, including the PSD (postsynaptic density) and membrane. The major PSD protein (mPSDp), constituting ϳ3% of its protein mass (and increasing rapidly after decapitation to 20% or more) was subsequently found to be CaMKII (Kennedy et al, 1983;Goldenring et al, 1984;Kelly et al, 1984).…”