Caspase-mediated Cleavage of the Ca2+/Calmodulin-dependent Protein Kinase-like Kinase Facilitates Neuronal Apoptosis

Marieke Kruidering,Theo Schouten,Gerard I Evan,Erno Vreugdenhil

doi:10.1074/jbc.m103471200

Marieke Kruidering, Theo Schouten + Show 2 more

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https://doi.org/10.1074/jbc.m103471200

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Abstract

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.

Highlights

This study was designed to identify the role of a recently identified Ca2؉/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis
CaMK-like1 kinase (CaMKLK) Is Expressed in NG108-15 Cells—To determine whether the novel kinase CaMKLK is expressed in NG108-15 cells, we performed Western blot analysis and RT-PCR experiments
In order to rule out the possibility of cross-reactivity, blots were stained with both anti-CaMKLK (Fig. 1A) and anti-CaMK-IV (Fig. 1B)

Summary

The abbreviations used are

CaMK, Ca2ϩ/calmodulin-dependent protein kinase; CaMKLK, CaMK-like kinase; CARP, CaMK-related peptide; CREB, cyclic AMP-responsive element-binding protein; DC, doublecortin; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PI, propidium iodide; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; DTT, dithiothreitol; JNK, c-Jun N-terminal kinase; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ZVAD.fmk, Z-Val-Ala-Asp(OMe)-CH2F; DEVD.fmk, Asp(OCH3)-Glu(OCH3)-Val(OCH3)-Asp(OCH3)-(OMe)-CH2F. A key factor in apoptosis is the activation of caspases, cysteine proteases that cleave a variety of proteins and by doing so disable important cellular processes and break down structural components such as laminin, eventually causing apoptotic cell death [13]. This, in combination with the fact that a CaMKLK splice variant is up-regulated during kainate-induced seizures, a process accompanied by apoptosis [23], prompted us to investigate the role of the CaMKLK gene in apoptosis. We found that it is a substrate for caspases and that overexpression of a noncleavable mutant protects NG108 cells from serum-deprived apoptosis, while overexpression of a caspase-generated serine/proline-rich N-terminal CaMKLK fragment exacerbates apoptosis. Our data suggest that cleavage of CAMKLK facilitates neuronal apoptosis

EXPERIMENTAL PROCEDURES

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DISCUSSION