Cloning and Characterization of a Protein Kinase A Anchoring Protein (AKAP)-related Protein That Interacts with and Regulates Sphingosine Kinase 1 Activity

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions. S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions. Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis. However, little is known of the mechanism(s) by which SPHK activity is regulated. Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP). BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger (ϳ7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP). In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not. Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated. Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions. The apoptoticsparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP. In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK. Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

“…2. Resolving the subtype selectivity of this AKAP is important in light of conflicting reports claiming that SKIP is an RII or RI anchoring protein (30,45,46).…”

“…SKIP was initially identified as a negative regulator of sphingosine kinase 1 (30). This enzyme is responsible for generation of sphingosine-1-phosphate (S1P), a cardioprotective and antiapoptotic lysophospholipid produced in response to hypoxia and acute ischemia/reperfusion injury (53).…”

“…SKIP was designated a PKA anchoring protein on the basis of homology with other AKAPs and its detection in a mass spectrometry screen for proteins enriched by purification on cAMP affinity resin (30,31). However, AKAPs are normally defined by their ability to interact directly with PKA (32).…”

“…5 F and H). Antipeptide antibodies against residues 44-65 of the anchoring protein were generated to detect endogenous SKIP in adult cardiac myocytes where this anchoring protein is enriched (30). Mouse SKIP was detected in two subcellular locations: a striated staining pattern reminiscent of Z bands and a distribution that aligns with mitochondrial marker proteins (Fig.…”

Sphingosine Kinase 2 Is a Nuclear Protein and Inhibits DNA Synthesis

“…SPHK1 predominantly localizes in the cytosol, and its overexpression induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (9). Several cellular proteins have recently been identified as SPHK1-interacting molecules, namely TRAF2 (10), RPK118 (11), and AKAP-related protein (12), which should help facilitate the understanding of the regulation and intracellular site of action of SPHK1.…”