One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin) another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed “SDK2”. An SDK separated as fractions “TN31−33” phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). (ii) The activation of these SDKs is specific to d- erythro-Sph and its N-methyl derivatives, the effect of l- threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. We showed previously that a purified SDK (termed “SDK1”) phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms β, η, and ζ but not τ or σ (Megidish, T., et al.
216, 739−747), have been termed “sphingosine-dependent kinases” (SDKs). Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N‘-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al.