Mitosis-specific Phosphorylation and Subcellular Redistribution of the RIIα Regulatory Subunit of cAMP-dependent Protein Kinase

Abstract

54 . Whereas RII␣ was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphaseanaphase transition. Furthermore, particulate RII␣ from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RII␣, and this may alter its subcellular localization.Cyclic AMP-dependent protein kinases (PKAs) 1 are present in mammalian tissues as two major isozymes, type I and type II (for reviews, see Refs 1 and 2). The inactive holoenzyme is composed of a regulatory subunit dimer that binds two catalytic subunits. Binding of cAMP to the regulatory subunits results in dissociation of the catalytic subunits, which are then catalytically active and can also be translocated to the nucleus.Four different regulatory subunits (RI␣, RI␤, RII␣, RII␤) and three different catalytic subunits (C␣, C␤, C␥) have been identified as separate gene products. The regulatory subunits RI␣ and RII␣ are present in almost all cell types, whereas the expression of RI␤ and RII␤ is tissue-specific. RII isoforms are associated with A kinase-anchoring proteins (AKAPs) (3) that target PKA type II to organelles, cytoskeleton, and membranes (3-5). Intracellular accumulation of RII has been observed in association with centrosome or the Golgi complex in several mammalian cell types (6 -8).The activities and subcellular localization of protein kinases and phosphatases are known to be regulated by phosphorylation (for a review, see Ref. 9). A major difference between RI and RII is the ability of RII to be autophosphorylated by the catalytic subunit of PKA (10). Phosphorylation sites for casein kinase II, glycogen synthase kinases 3 and 5, and prolinedirected protein kinase have been identified in vitro on bovine RII␣ as well (11-13). More recently it was shown that bovine RII␤ can also be phosphorylated at Thr 69 in vitro by CDK1, the mitotic kinase p34 cdc2 associated with cyclinB (14). The physiological role of these phosphorylations is not fully understood. In vitro autophosphorylation of PKA type II decreases the affinity between RII and the catalytic subunit, and CDK1 phosphorylation of RII␤ decreases its ability to anchor to MAP-2 (a neuronal microtubule-associated protein (14)). However, RII␤ is expressed primarily in neuro-endocrine cells and is thus present in differentiated cells that divide slowly or not at all. For these reasons, we examined the cell cycle-dependent phosphorylation of the ubiquitously expressed RII␣. In the present study, we show that in dividing cells, the regulatory subunit RII␣ demonstrates different phosphorylation patterns during the cell cycle. In mitotic-arrested cells, RII␣-labeling with 8-azido[ 32 P]cAMP, immunostaining with a specific antibody, and in situ incorporation of [ 32 P]orthophosphate in HeLa cells revealed different states of RII␣ phosphorylation compared with that observed during interphase. Inhibition of PKA activity with a selective inhibitor, H89, or...

Modulation of the cAMP signaling pathway after traumatic brain injury

“…cAMP primarily exerts its actions through PKA. When PKA is activated, the catalytic subunit autophosphorylates the regulatory subunit and this may facilitate dissociation and activation of the catalytic subunits (Keryer et al, 1998). To determine whether PKA activation is modulated after TBI, we performed western blot analysis with antibodies to phosphorylated, activated PKA regulatory subunit II.…”

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…Strikingly, in the presence of U0126, late G 2 cells exhibited linked rather than unlinked Golgi elements, and the number of discrete elements matched control values (Figure 7, A-C, late G 2 ϩ U0126), indicating that MEK1 activity is required for Golgi unlinking in late G 2 . Furthermore, olomoucine II treatment, which induced the expected arrest in late G 2 (Keryer et al, 1998) was also observed in a distinct cell type. Synchronized normal rat kidney (NRK) cells were analyzed using anti-giantin immunostaining, and the G 2 population of cells exhibited a significant increase in unlinked Golgi elements compared with the S-phase population, but this increase was absent if U0126 was added to inhibit MEK activity ( Figure 7D).…”

“…As expected, the Golgi ribbon was intact in S-phase cells, and green fluorescent protein (GFP)-labeled GalNacT2 rapidly recovered after photobleaching (Figure 6, A, C, and Supplemental Figure S3 movie). To accumulate late G 2 cells, thymidine washout was carried out in the presence of the CDK1 inhibitor olomoucine II (Krystof et al, 2005), which induced the expected arrest just before M phase (Keryer et al, 1998). Importantly, in late G 2 cells the ribbon was significantly fragmented (quantified below), and there was impaired fluorescence recovery even when the photobleaching was carried out on a region in proximity to other Golgi structures ( Figure 6, B and C, and Supplemental Figure S4 movie).…”

Involvement of membrane GRP78 in trophoblastic cell fusion

“…Trophoblastic cells fuse in response to treatment with cAMP [7] [19]. Forskolin is often used to induce cAMP level and fusion of trophoblastic cells [19].…”