Characterization of the protein kinases in a transplantable islet cell tumor of the syrian hamster

Abstract

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70–80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I ( M r 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R 2 ( M r 90,000) and a catalytic subunit C ( M r 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg 2+ and ATP. The regulatory subunit, R 2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R 1 ( M r 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase (Beavo et al., 1974), an R 2C 2 structure is proposed for the cAMP-dependent insulinoma protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R 2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase ( M r 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II ( M r 88,000), is the predominant form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protamine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same M r (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.