ABSTRACT
The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.
Subject(s)
Muscle Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Protein Kinases/metabolism , Animals , Binding Sites , Brain/enzymology , Casein Kinases , Catalytic Domain , Humans , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Phosphoprotein Phosphatases , Phosphorylation , Protein Kinase C/chemistry , Protein Kinases/chemistry , Protein Structure, TertiaryABSTRACT
Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated. The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast. Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive. We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin. In this report, we have focused on the interaction of centaurin-alpha(1) with PKC. All groups of PKC associate directly through their cysteine rich domains. Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation. This is the first report of a kinase that phosphorylates centaurin-alpha(1).
Subject(s)
Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Kinase C/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Carrier Proteins/chemistry , Isoenzymes/metabolism , Nerve Tissue Proteins/chemistry , Phosphorylation , Protein Kinase C/chemistry , Protein Structure, TertiaryABSTRACT
Centaurin-alpha(1) was originally described as a binding partner for phosphoinositides. In spite of the presence of a putative ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain, no ARF-GAP activity has been attributed to centaurin-alpha(1) so far. Thus the function of this protein remains to be determined. In order to better understand its intracellular role, we aimed to identify centaurin-alpha(1) partners. Using affinity chromatography followed by mass spectrometry analysis, we identified several potential centaurin-alpha(1) protein partners. Nucleolin, a nucleolar protein involved in ribosome biosynthesis, was the main centaurin-alpha(1) interacting protein. The interaction between centaurin-alpha(1) and nucleolin was confirmed by Western blot analysis and GST pull down assays. Moreover, we have shown that ectopically expressed centaurin-alpha(1) associates in vivo with endogenous nucleolin in human embryonic kidney 293 cells. In addition, the association between nucleolin and centaurin-alpha(1) was disrupted by RNAse treatment, indicating that RNA integrity was necessary for their binding. This suggested that centaurin-alpha(1) was part of a ribonucleoprotein complex.
Subject(s)
Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Brain Chemistry , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Fractionation , Cell Line , Chromatography, Affinity , Humans , Immunohistochemistry , Mass Spectrometry , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Phosphoproteins/chemistry , Protein Binding , RNA/metabolism , RNA-Binding Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sheep , NucleolinABSTRACT
CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.
Subject(s)
Brain/metabolism , Muscle Proteins/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoproteins/metabolism , Brain Chemistry , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Muscle Proteins/isolation & purification , Muscle Proteins/pharmacology , Phosphoprotein Phosphatases/metabolism , Phosphoproteins/isolation & purification , Phosphoproteins/pharmacology , Phosphorylation , Protein Phosphatase 1 , Serine/metabolism , Threonine/metabolismABSTRACT
Casein kinase Ialpha (CKIalpha) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIalpha interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin-alpha1, synaptotagmin IX and CPI-17 that were isolated from brain as CKIalpha co-purifying proteins. Actin, importin-alpha(1), importin-beta, PP2Ac, centaurin-alpha1, and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214-233 of CKIalpha. We have previously shown that centaurin-alpha1 represents a CKIalpha partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIalpha and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217-233 of CKIalpha which are proposed from X-ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIalpha complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIalpha protein partners and their relations to CKI are discussed.
