Toolsee J. Singh

Potentiation of GSK-3-catalyzed Alzheimer-like phosphorylation of human tau by cdk5

Tau protein from Alzheimer disease (AD) brain is hyperphosphorylated by both proline-dependent protein kinases (PDPKs) and non-PDPKs. It is presently unclear how PDPKs and non-PDPKs interact in tau hyperphosphorylation. Previously we have shown that non-PDPKs can positively modulate the activity of a PDPK (GSK-3) in tau phosphorylation (Singh et al. (1995) FEBS Lett. 358, 267-272). In this study we have investigated whether (A) non-PDPKs can also modulate the activity of the PDPK, cdk5, (B) a PDPK can modulate the activities of another PDPK, as well as non-PDPKs. We found that, like GSK-3, the activity of cdk5 is stimulated if tau were first prephosphorylated by any of several non-PDPKs (A-kinase, C-kinase, CK-1, CaM-kinase II). Prephosphorylation of tau by cdk5 stimulated both the rate and extent of a subsequent phosphorylation catalyzed by GSK-3. Under these conditions thr 231 phosphorylation was especially enhanced (9-fold). No significant stimulation of phosphorylation was obser ved when the order of these kinases was reversed (i.e. GSK-3 followed by cdk5). By contrast, prephosphorylation of tau by cdk5 served to inhibit subsequent phosphorylation catalyzed by C-kinase and CK-1, but not by A-kinase or CaM-kinase II. Our results suggest that in tau hyperphosphorylation in AD brain, cdk5-catalyzed phosphorylation may serve to up-regulate the activity of GSK-3 and down-regulate the activities of C-kinase and CK-1. (Mol Cell Biochem 167: 99-105, 1997)

MODULATION OF GSK-3-CATALYZED PHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN TAU BY NON-PROLINE-DEPENDENT PROTEIN KINASES

The phosphorylation of bovine tau, either by GSK‐3 alone or by a combination of GSK‐3 and several non‐proline‐dependent protein kinases (non‐PDPKs), was studied. GSK‐3 alone catalyzed the incorporation of ∼ 3 mol 32P/mot tau at a relatively slow rate. Prephosphorylation of tau by A‐kinase, C‐kinase, or CK‐2 (but not by CK‐1, CaM kinase II or Gr kinase) increased both the rate and extent of a subsequent phosphorylation catalyzed by GSK‐3 by several‐fold. These results suggest that the phosphorylation of tau by PDPKs such as GSK‐3 (and possibly MAP kinase, cdk5) may be positively modulated at the substrate level by non‐PDPK‐catalyzed phosphorylations.

Rapid Alzheimer-like phosphorylation of tau by the synergistic actions of non-proline-dependent protein kinases and GSK-3

Tau protein from Alzheimer disease (AD) brain is phosphorylated at eleven Ser/Thr‐Pro and nine Ser/Thr‐X sites. The former sites are phosphorylated by proline‐dependent protein kinases (PDPKs), the latter by non‐PDPKs. The identities of both the PDPKs and non‐PDPKs involved in AD tau hyperphosphorylation are still to be established. In this study we have analyzed the interactions between a PDPK (GSK‐3) and several non‐PDPKs (A‐kinase, C‐kinase, CK‐1, CaM kinase II) in the phosphorylation of one isoform (tau 39) of human tau. We found that the rate of phosphorylation of tau 39 by GSK‐3 was increased several‐fold if tau were first prephosphorylated by the non‐PDPKs. Further, several Alzheimer‐like epitopes in tau can be induced only slowly after phosphorylation of tau by GSK‐3 alone. After a prephosphorylation of tau by the non‐PDPKs, however, the rate of induction of these epitopes by GSK‐3 is increased several‐fold. These results suggest that one role of non‐PDPK‐catalyzed phosphorylation is the modulation of PDPK‐catalyzed phosphorylation of tau in AD brain.

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization

The distribution of glycogen synthase (casein) kinase‐1 (CK‐1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK‐1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK‐1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK‐1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK‐1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK‐1 may be involved in the regulation of diverse cellular functions.

Protein kinase C and calcium/calmodulin-dependent protein kinase II phosphorylate three-repeat and four-repeat tau isoforms at different rates

All six isoforms of the microtubule-associated protein tau are present in hyperphosphorylated states in the brains of patients with Alzheimers disease (AD). It is presently unclear how such hyperphosphorylation of tau is controlled. In a previous study (Singh et al. Arch Biochem Biophys 328: 43-50, 1996) we have shown that three-repeat taus containing two N-terminal inserts were phosphorylated to higher levels and at different sites compared to those either lacking or containing only one such insert. We have extended these observations in this study by comparing the phosphorylation of tau isoforms containing three-repeats (t3, t3L) and four-repeats (t4, t4L). In the absence of N-terminal inserts in tau structure (t3, t4) both CaM kinase II and C-kinase phosphorylated four-repeat tau (t4) to a higher extent than three-repeat tau (t3). When two N-terminal inserts are present in tau structure (t3L, t4L), then three-repeat tau (t3L) is phosphorylated to a higher extent than four-repeat tau (t4L) by these kinases. CK-1 and GSK-3 phosphorylated each of the above pairs of three-repeat and four-repeat taus to the same extents. However, after an initial prephosphorylation of the taus by CaM kinase II, GSK-3 differentially phosphorylated three-repeat and four-repeat taus. Under these conditions thr 231, ser 235, ser 396, and ser 404 were phosphorylated to greater extents in four-repeat tau (t4) compared to three-repeat tau (t3) in the absence of N-terminal inserts. In the presence of such inserts these sites were phosphorylated to greater extents in three-repeat (t3L) compared to four-repeat (t4L) tau. Our results indicate that the extents to which tau isoforms are phosphorylated in normal and AD brain depends on (a) the number of repeats (3 or 4), (b) the number of N-terminal inserts (0, 1, or 2), and (c) the initial phosphorylation state of tau.

Phosphorylation of troponin and myosin light chain by cAMP-independent casein kinase-2 from rabbit skeletal muscle

Casein kinase-2 from rabbit skeletal muscle was found to phosphorylate, in addition to glycogen synthase, troponin from skeletal muscle, and myosin light chain from smooth muscle. Troponin T and the 20,000 Mr myosin light chain are phosphorylated by casein kinase-2 at much greater rates than glycogen synthase. The V values for the phosphorylation of troponin and myosin light chain are nearly an order of magnitude greater than that of glycogen synthase; however, the Km values for these two substrates are greater than that for glycogen synthase. The kinase activities with the various protein substrates are stimulated approximately three- and fivefold by 5 mM spermidine and 3 mM spermine, respectively. Heparin is a potent inhibitor of the kinase when casein, glycogen synthase, or myosin light chain is the substrate. However, with troponin as substrate the kinase is relatively insensitive to inhibition by heparin. The amount of heparin required for 50% inhibition with troponin as substrate is at least 10 times greater than with casein as substrate. The phosphorylation of troponin by casein kinase-2 results in the incorporation of phosphate into two major tryptic peptides, which are different from those phosphorylated by casein kinase-1. The site in myosin light chain phosphorylated by casein kinase-2 is different from that phosphorylated by myosin light chain kinase.

Phosphorylation of smooth muscle myosin light chain by five different kinases

Phosphorylation of the 20 kDa myosin light chain from smooth muscle by five different kinases was investigated. Three of the kinases (myosin light chain kinase, phosphorylase kinase, and cAMP‐dependent protein kinase) phosphorylate serine residues, the fourth (casein‐kinase‐2) mainly threonine, and the fifth (glycogen synthase (casein) kinase‐1) both serine and threonine. Isoelectric focusing analyses of 32P‐labelled chymotryptic peptides indicate that phosphorylase kinase and cAMP‐dependent protein kinase phosphorylate the same site as myosin light chain kinase. However, both casein kinase‐2 and glycogen synthase (casein) kinase‐1 phosphorylate different sites.

Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1.

MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

Abstract A cyclic nucleotide- and Ca2+-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. 254 , 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca2+-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.