Calcium/Calmodulin-dependent Protein Kinase IV Is Cleaved by Caspase-3 and Calpain in SH-SY5Y Human Neuroblastoma Cells Undergoing Apoptosis

Abstract

We have previously demonstrated cleavage of ␣-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the proapoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxyAsp-CH 2 OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Aspfluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD 176 *A and CG 201 *A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules

“…CaMK IV but not CaMK IIb is rapidly cleaved during cell death with kinetics that precede the observed decrease in CaMK activity. 74 In addition to CaMK IV, CaMK IIa and the upstream activator CaMK kinase (CaMKK) are also cleaved in vivo in SH-SY5Y cells exposed to staurosporine, a finding that is reminiscent of the observed sequential cleavage of several MAPK components such as MEKK-1 and MSTs. CaMK IV is also cleaved by calpain to a 40 kD fragment in a manner that is inhibited by a calpain inhibitor, both in vivo and in vitro.…”

“…5 CaMK IV is a serine-threonine kinase with restricted tissue distribution and a high degree of homology to CaMK II. 74 CaMK IV has been implicated in activation of a number of important transcription factors (Table 2). Like the situation for other kinases such as DNA-PK and PKCz, inhibition of CaMK kinase activity is associated with apoptosis.…”

“…71,72 Calpain is known to cleave other signaling molecules during apoptosis, including CamK IV and PKCy. 73,74 (vii) Resetting the apoptotic switch through RNA splicing of apoptosis regulatory molecules: changing the rules of battle after the battle starts…”

Neuronal loss in primary long-term cortical culture involves neurodegeneration-like cell death via calpain and p35 processing, but not developmental apoptosis or aging

“…The two major calpain family proteins, µ-calpain and m-calpain, share a 30 kDa regulatory subunit and require different Ca 2+ concentrations for activation in vitro. Activated calpain acts on a host of endogenous proteins, which include cytoskeletal proteins (fodrin, vimentin, keratin, and tau), PARP, calmodulin-dependent protein kinase IV (CaMKIV), clathrin assembly protein, bax, and even procaspase-3 (Ando et al, 1988;Litersky and Johnson, 1995;McGinnis et al, 1998;Prasad et al, 1998;McGinnis et al, 1999a;Kim and Kim, 2001). Particularly, in many neurodegenerative disorders such as Alzheimer's disease, this calpain systems are involved (Patrick et al, 1999;Lee et al, 2000).…”

Exploiting an NQ01-Directed, Calpain-Medicated Apoptotic Pathway for Breast Cancer Therapy

“…Calpains can also inactivate caspases by cleavage of the pro-caspase form to proteolytically inactive fragments (52,79). In addition, calpain activation in apoptosis can occur concurrently with, or in the absence of, caspase activation (80)(81)(82)(83)(84)(85)(86). In studies described above, we demonstrated that calpains were activated in human breast cancer cells during ß -lap-mediated apoptosis, while caspase activation was not apparent (Fig.…”