Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP.

Schulman, Howard

doi:10.1128/mcb.4.6.1175

Cited by 42 publications

(8 citation statements)

References 28 publications

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“…The demonstration in this report that microtubule preparations contain an endogenous calmodulin-dependent MAP 2 kinase indicates that both calmodulin-and cAMP-dependent kinases may play a role in regulating microtubule function. A previous report indicated that a purified calmodulin-dependent kinase phosphorylated distinct sites on MAP 2 compared to cAMP-dependent kinase (26). In the present report, the endogenous calmodulindependent kinase in the void fraction incorporated at least 5 mol of phosphate per mol of endogenous MAP 2.…”

Section: Resultssupporting

confidence: 58%

Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.

Vallano¹,

Goldenring²,

Buckholz³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Both cAMP-and calmodulin-dependent kinases are proposed regulators of microtubule function by means of their ability to phosphorylate microtubule-associated protein 2 (MAP 2). A cAMP-dependent kinase/MAP 2 complex is endogenous to microtubules. In this report, we demonstrate that an endogenous calmodulin-dependent kinase that phosphorylates MAP 2 as a major substrate is also present in microtubules prepared under conditions that preserve kinase activity. This enzyme is identical to a calmodulin-dependent kinase purified previously from rat brain cytosol. A fraction containing calmodulin-dependent kinase and MAP 2 was separated from the cAMP-dependent kinase/MAP 2 complex by gel fltration chromatography ofmicrotubule protein in high ionic strength buffer. AU of the recovered calmodulin-dependent kinase activity in microtubules eluted in a single protein peak. The specific activity of the enzyme for MAP 2 was enriched 31-fold in this fraction compared to cytosol. Twodimensional tryptic phosphopeptide mapping revealed that the endogenous cAMP-and calmodulin-dependent kinases phosphorylated distinct sites on MAP 2. These data demonstrate that both kinases are present in microtubule preparations and that they may differentially regulate MAP 2 function by phosphorylating separate sites on MAP 2.Microtubules play a pivotal role in a variety of dynamic intracellular processes. In situ and in vitro studies indicate that microtubules are crosslinked to one another, neurofilaments and other intermediate filaments, actin, and secretory vesicles through the high molecular weight microtubule-associated protein 2 (MAP 2) (for review, see ref. 1). Rapid modulation of cytoskeletal function may be effected by reversible phosphorylation of MAP 2. MAP 2 is phosphorylated in vivo (2) and reconstitution studies indicate that phosphorylation of MAP 2 with cAMP-dependent kinase (3) or calmodulin-dependent kinase (4) diminishes its ability to promote tubulin assembly into microtubules. A cAMPdependent kinase activity is endogenous to microtubule preparations and phosphorylates 8-13 of the 20-22 available sites on MAP 2 (1).

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Section: Resultssupporting

confidence: 58%

Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.

Vallano¹,

Goldenring²,

Buckholz³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…There is very little data on the precise location of endogenous phosphorylation sites in MAP2 . Both cAMP-dependent and Cal+/calmodulin kinases have been shown to phosphorylate sites in the portion of the molecule that is bound to microtubules (Vallee, 1980;Schulman, 1984 ;Vallano et al ., 1985 ;Hernandez et al, 1987) . However, it is clear that the degree of phosphorylation of all in vitro preparations is less than that of MAP2 in the living brain (Tsuyama et al, 1987), and other phosphorylation sites might exist that also influence the interaction of MAP2 with microtubules .…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells.

Brugg

Matus

1991

The Journal of Cell Biology

203

109

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“…Indeed, in vitro studies have shown that the phosphorylation of MAPs prevents them from functioning to promote microtubule assembly, thus, resulting in disassembly or increased flux of microtubules (16,17). Furthermore, Schulman (34) has reported that the phosphorylation of MAP-2 is stimulated by a Ca2÷-CaM-dependent kinase in vitro. Thus, a precedent for such a mechanism in the mitotic spindle exists.…”

Section: Discussionmentioning

confidence: 99%

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

Brady¹,

Cabral²,

Dedman³

1986

The Journal of Cell Biology

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Abstract. A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinitypurified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.

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Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP.

Cited by 42 publications

References 28 publications

Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.

Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.

Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells.

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

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