Balu Chakravarthy

Ca2+–calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains

Evidence is accumulating that suggests that Ca2+-calmodulin (Ca2+-CaM) and the protein kinase Cs (PKCs) obstruct each others actions because of the embedding of PKC phosphorylation sites in CaM or Ca2+-CaM-binding domains of a growing number of crucial substrates in neurons (and other cells). These substrates include the CaM storage proteins (neurogranin, neuromodulin), the membrane-associated MARCKS (myristoylated alanine-rich C-kinase substrate) protein, the NMDA receptor RI subunit and the autoinhibitory domain of the plasma membrane Ca2+ pump. In this review, the emerging data are woven into a hypothetical picture of the conflicting, timing-dependent convergence of two major signalers on neuronal functions.

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.

Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons

Abstract: A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15–20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20–50 µM glutamate for 15 min was neurotoxic and induced a rapid (∼1–2 h) Ca2+‐dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to >800 µM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription‐polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2+ concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptor on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (<20 µM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 µM calphostin C, 5 µM chelerythrine, or chronic exposure to 100 nM PMA. A 15‐min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate‐mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate‐mediated neuronal death.

Parathyroid hormone fragment [3-34] stimulates protein kinase C (PKC) activity in rat osteosarcoma and murine T-lymphoma cells.

The parathyroid hormone (PTH) fragment [1-34] strongly stimulated both adenylate cyclase and membrane-associated PKC activities in rat 17/2 osteosarcoma cells. By contrast, the PTH [3-34] fragment, which was unable to stimulate adenylate cyclase, remained a potent stimulator of membrane-associated PKC activity in these cells. Both PTH fragments also strongly stimulated membrane-PKC activity in cyc-S49T-lymphoma cells possessing a defective adenylate cyclase system. This ability of PTH [3-34] to stimulate membrane-associated PKC activity could explain the residual bioactivity of this fragment.

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

An early loss in membrane protein kinase C activity precedes the excitatory amino acid-induced death of primary cortical neurons.

Abstract: Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15‐min exposure of day 14–18 cortical neurons to 100 µM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30–50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC‐selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral ischemia in vivo.

Stimulation of Protein Kinase C during Ca-induced Keratinocyte Differentiation SELECTIVE BLOCKADE OF MARCKS PHOSPHORYLATION BY CALMODULIN

Raising the external Ca concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca- or TPA-stimulated keratinocytes. Raising the external Ca concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. LopezBarahona, M., and Aaronson, S. A.(1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca did not. Furthermore, Ca-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca•CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca. Ca•calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca•calmodulin complexes, formed by the internal Ca surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.

Calpain-mediated truncation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H2O2 toxicity

Dihydropyrimidinase‐like protein 3 (DPYSL3), a member of TUC (TOAD‐64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and, possibly, neuronal regeneration. In primary cortical cultures, glutamate (NMDA) excitotoxicity and oxidative stress (H2O2) caused the cleavage of DPYSL3, resulting in the appearance of a doublet of 62 kDa and 60 kDa. Pre‐treatment of cell cultures with calpain inhibitors, but not caspase 3 inhibitor, before exposure to NMDA or H2O2 completely blocked the appearance of the doublet, suggesting calpain‐mediated truncation. Furthermore, in vitro digestion of DPYSL3 in cell lysate with purified calpain revealed a cleavage product identical to that observed in NMDA‐ and H2O2‐treated cells, and its appearance was blocked by calpain inhibitors. Analysis of the DPYSL3 protein sequence revealed a possible cleavage site for calpain (Val‐Arg‐Ser) on the C‐terminus of DPYSL3. Collectively, these studies demonstrate for the first time that DPYSL3 is a calpain substrate. The physiological relevance of the truncated DPYSL3 protein remains to be determined.

Activation of DNA‐Dependent Protein Kinase May Play a Role in Apoptosis of Human Neuroblastoma Cells

Abstract : Treating SH‐SY5Y human neuroblastoma cells with 1 μM staurosporine resulted in a three‐ to fourfold higher DNA‐dependent protein kinase (DNA‐PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA‐PK activity : an initial increase that peaked by 4 h and a rapid decline that reached ~5‐10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA‐PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase‐1 activity but stimulated caspase‐3 activity by 10‐15‐fold in a time‐dependent manner, similar to morphological changes. Similar time‐dependent changes in DNA‐PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 μM ceramide or UV radiation. In all these cases the increase in DNA‐PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell‐permeable inhibitor of DNA‐PK, OK‐1035, significantly reduced staurosporine‐induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA‐PK may be involved with the induction of apoptotic cell death.

C-terminal fragment of parathyroid hormone-related protein, PTHrP-(107-111), stimulates membrane-associated protein kinase C activity and modulates the proliferation of human and murine skin keratinocytes.

Low concentrations of the C‐terminal parathyroid hormone‐related protein (PTHrP) fragments, PTHrP‐(107–111) and PTHrP‐(107–139), stimulated membrane‐associated protein kinase Cs (PKCs), but not adenylyl cyclase or an internal Ca2+ surge, in early passage human skin keratinocytes and BALB/MK‐2 murine skin keratinocytes. The fragment maximally stimulated membrane‐associated PKCs in BALB/MK‐2 cells at 5 × 10−9 to 10−8 M. The maximally PKC‐stimulating concentrations of PTHrP‐(107–111) also stopped or stimulated BALB/MK‐2 keratinocyte proliferation depending on whether the cells were, respectively, cycling or quiescent at the time of exposure. Thus, just one brief (30‐minute) pulse of 10 −8 M PTHrP‐(107–111) stopped the proliferation of BALB/MK‐2 keratinocytes for at least 5 days. On the other hand, daily 30‐minute pulses of 10−8 M PTHrP‐(107–111) started and then maintained the proliferation of initially quiescent BALB/MK‐2 cells. Similarly PTHrP‐(107–111) inhibited DNA synthesis by cycling primary adult human keratinocytes, but it stimulated DNA synthesis by quiescent human keratinocytes.